Mesenchymal stem cell – Wikipedia

Medicinal signaling cells (MSCs) previously known as Mesenchymal stem cells are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells which give rise to marrow adipose tissue).[1][2]

While the terms mesenchymal stem cell (MSC) and marrow stromal cell have been used interchangeably for many years, neither term is sufficiently descriptive:

Mesenchymal stem cells are characterized morphologically by a small cell body with a few cell processes that are long and thin. The cell body contains a large, round nucleus with a prominent nucleolus, which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear appearance. The remainder of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria and polyribosomes. The cells, which are long and thin, are widely dispersed and the adjacent extracellular matrix is populated by a few reticular fibrils but is devoid of the other types of collagen fibrils.[7][8]

Bone marrow was the original source of MSCs, and still is the most frequently utilized. These bone marrow stem cells do not contribute to the formation of blood cells and so do not express the hematopoietic stem cell marker CD34. They are sometimes referred to as bone marrow stromal stem cells.[9]

The youngest and most primitive MSCs may be obtained from umbilical cord tissue, namely Wharton's jelly and the umbilical cord blood. However MSCs are found in much higher concentration in the Whartons jelly compared to cord blood, which is a rich source of hematopoietic stem cells. The umbilical cord is available after a birth. It is normally discarded and poses no risk for collection. These MSCs may prove to be a useful source of MSCs for clinical applications due to their primitive properties.

Adipose tissue is a rich source of MSCs (or adipose-derived mesenchymal stem cells, AdMSCs).[10]

The developing tooth bud of the mandibular third molar is a rich source of MSCs. While they are described as multipotent, it is possible that they are pluripotent. They eventually form enamel, dentin, blood vessels, dental pulp and nervous tissues. These stem cells are capable of producing hepatocytes.

Stem cells are present in amniotic fluid. As many as 1 in 100 cells collected during amniocentesis are pluripotent mesenchymal stem cells.[11]

MSCs have a great capacity for self-renewal while maintaining their multipotency. Recent work suggests that -catenin, via regulation of EZH2 , is a central molecule in maintaining "stemness" of MSC's.[12] The standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes and chondrocytes as well as myocytes.

MSCs have been seen to even differentiate into neuron-like cells,[13] but doubt remains about whether the MSC-derived neurons are functional.[14] The degree to which the culture will differentiate varies among individuals and how differentiation is induced, e.g., chemical vs. mechanical;[15] and it is not clear whether this variation is due to a different amount of "true" progenitor cells in the culture or variable differentiation capacities of individuals' progenitors. The capacity of cells to proliferate and differentiate is known to decrease with the age of the donor, as well as the time in culture. Likewise, whether this is due to a decrease in the number of MSCs or a change to the existing MSCs is not known.[citation needed]

MSCs have an effect on innate and specific immune cells. MSCs produce many molecules having immunomodulatory effects. These include prostaglandin E2 (PGE2),[16] nitric oxide,[17] indolamin 2,3-dioxigenase (IDO), IL-6, and other surface markers - FasL,[18] PD-L1 / 2.

MSCs have an effect on macrophages, neutrophils, NK cells, mast cells and dendritic cells in innate immunity. MSCs are able to migrate to the site of injury, where they polarize through PGE2 macrophages in M2 phenotype which is characterized by an anti-inflammatory effect.[19] Further, PGE2 inhibits the ability of mast cells to degranulate and produce TNF-.[20][21] Proliferation and cytotoxic activity of NK cells is inhibited by PGE2 and IDO. MSCs also reduce the expression of NK cell receptors - NKG2D, NKp44 and NKp30.[22] MSCs inhibit respiratory flare and apoptosis of neutrophils by production of cytokines IL-6 and IL-8.[23] Differentiation and expression of dendritic cell surface markers is inhibited by IL-6 and PGE2 of MSCs.[24] The immunosuppressive effects of MSC also depend on IL-10, but it is not certain whether they produce it alone, or only stimulate other cells to produce it.[25]

MSC expresses the adhesion molecules VCAM-1 and ICAM-1, which allow T-lymphocytes to adhere to their surface. Then MSC can affect them by molecules which have a short half-life and their effect is in the immediate vicinity of the cell.[17] These include nitric oxide,[26] PGE2, HGF,[27] and activation of receptor PD-1.[28] MSCs reduce T cell proliferation between G0 and G1 cell cycle phases[29] and decrease the expression of IFN of Th1 cells while increasing the expression of IL-4 of Th2 cells.[30] MSCs also inhibit the proliferation of B-lymphocytes between G0 and G1 cell cycle phases.[28][31]

MSCs can produce antimicrobial peptides (AMPs). These include human cathelicidin LL-37,[32] -defensines,[33] lipocalin 2[34] and hepcidin.[35] MSCs effectively decrease number of colonies of both gram negative and gram positive bacteria by production of these AMPs. In addition, the same antimicrobial effect of the enzyme IDO produced by MSCs was found.[36]

Mesenchymal stem cells in the body can be activated and mobilized if needed. However, the efficiency is low. For instance, damage to muscles heals very slowly but further study into mechanisms of MSC action may provide avenues for increasing their capacity for tissue repair.[37][38]

Clinical studies investigating the efficacy of mesenchymal stem cells in treating diseases are in preliminary development, particularly for understanding autoimmune diseases, graft versus host disease, Crohn's disease, multiple sclerosis, systemic lupus erythematosus and systemic sclerosis.[39][40] As of 2014, no high-quality clinical research provides evidence of efficacy, and numerous inconsistencies and problems exist in the research methods.[40]

Many of the early clinical successes using intravenous transplantation came in systemic diseases such as graft versus host disease and sepsis. Direct injection or placement of cells into a site in need of repair may be the preferred method of treatment, as vascular delivery suffers from a "pulmonary first pass effect" where intravenous injected cells are sequestered in the lungs.[41]

The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define MSCs. A cell can be classified as an MSC if it shows plastic adherent properties under normal culture conditions and has a fibroblast-like morphology. In fact, some argue that MSCs and fibroblasts are functionally identical.[42] Furthermore, MSCs can undergo osteogenic, adipogenic and chondrogenic differentiation ex vivo. The cultured MSCs also express on their surface CD73, CD90 and CD105, while lacking the expression of CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR surface markers.[43]

The majority of modern culture techniques still take a colony-forming unit-fibroblasts (CFU-F) approach, where raw unpurified bone marrow or ficoll-purified bone marrow Mononuclear cell are plated directly into cell culture plates or flasks. Mesenchymal stem cells, but not red blood cells or haematopoetic progenitors, are adherent to tissue culture plastic within 24 to 48 hours. However, at least one publication has identified a population of non-adherent MSCs that are not obtained by the direct-plating technique.[44]

Other flow cytometry-based methods allow the sorting of bone marrow cells for specific surface markers, such as STRO-1.[45] STRO-1+ cells are generally more homogenous and have higher rates of adherence and higher rates of proliferation, but the exact differences between STRO-1+ cells and MSCs are not clear.[46]

Methods of immunodepletion using such techniques as MACS have also been used in the negative selection of MSCs.[47]

The supplementation of basal media with fetal bovine serum or human platelet lysate is common in MSC culture. Prior to the use of platelet lysates for MSC culture, the pathogen inactivation process is recommended to prevent pathogen transmission.[48]

New research titled Transplantation of human ESC-derived mesenchymal stem cell spheroids ameliorates spontaneous osteoarthritis in rhesus macaques[49]

In 1924, Russian-born morphologist Alexander A. Maximov (Russian: ); used extensive histological findings to identify a singular type of precursor cell within mesenchyme that develops into different types of blood cells.[50]

Scientists Ernest A. McCulloch and James E. Till first revealed the clonal nature of marrow cells in the 1960s.[51][52] An ex vivo assay for examining the clonogenic potential of multipotent marrow cells was later reported in the 1970s by Friedenstein and colleagues.[53][54] In this assay system, stromal cells were referred to as colony-forming unit-fibroblasts (CFU-f).

The first clinical trials of MSCs were completed in 1995 when a group of 15 patients were injected with cultured MSCs to test the safety of the treatment. Since then, more than 200 clinical trials have been started. However, most are still in the safety stage of testing.[5]

Subsequent experimentation revealed the plasticity of marrow cells and how their fate is determined by environmental cues. Culturing marrow stromal cells in the presence of osteogenic stimuli such as ascorbic acid, inorganic phosphate and dexamethasone could promote their differentiation into osteoblasts. In contrast, the addition of transforming growth factor-beta (TGF-b) could induce chondrogenic markers.[citation needed]

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Mesenchymal stem cell - Wikipedia

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Human mesenchymal stem cells – current trends and future …

Abstract

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term invitro culturing, invitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.

Keywords: chronic diseases, homing, immunomodulatory features, in vitro differentiation, mesenchymal stem cells

Abbreviations: AD, Alzheimer disease; AD-MSC, adipose-derived mesenchymal stem cell; ALS, amylotrophic lateral sclerosis; BDNF, brain-derived neurotrophic factor; BME, -mercaptoethanol; BM-MSC, bone marrow-derived mesenchymal stem cell; BMP, bone morphogenic protein; CD, cluster of differentiation; CPA, cryoprotective agent; CRF, controlled rate freezer; DA, dopamine; DMEM, Dulbecco's modified Eagle's media; EGF, epidermal growth factor; ESC, embryonic stem cell; FCS, fetal calf serum; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HLA, human leucocyte antigen; hMSC, human mesenchymal stem cell; ICM, inner cell mass; IFN, interferon; IL, interleukin; IMDM, Iscove's modified Dulbecco's medium; iPSC, induced pluripotent stem cell; LMX1a, LIM homoeobox transcription factor 1 ; MHC, major histocompatibility complex; MMP, matrix metallo-protease; MSC, mesenchymal stem cell; NBCS, new-born calf serum; NK, natural killer; PD, Parkinson's disease; PD, population doubling; PPAR, peroxisome proliferator-activated receptor ; RA, rheumatoid arthritis; Runx2, runt-related transcription factor 2; SSEA, stage-specific embryonic antigen; TGF-, transforming growth factor-; Th, T helper cell; TLR, toll-like receptor; Treg, regulatory T-cell; UCB-MSC, umbilical cord blood-derived mesenchymal stem cell

In this review, we highlighted recent research findings in the area of human mesenchymal stem cells, its application in the treatment of chronic diseases and its use in human clinical trials.

Stem cells are the cells with a specific function with the ability of self-renewal, possess varied potency and differentiate into multilineages [1]. Because of clinical applications and biological importance, stem cells become a prominent subject in modern research era. On the basis of origin, stem cells are divided into different categories.

Embryonic stem cells (ESCs) are pluripotent stem cells, isolated originally from the inner cell mass (ICM) of mouse early pre-implantation blastocyst, having the capacity to generate into any mature cell of the three germ lines [2]. Later on, Thomson et al. [3] also isolated ESCs from ICM of human blastocyst, but until now as compared with humans, only mouse ESCs have been investigated in depth. ESCs possess distinctive self-renewal capacity, pluripotency and genomic stability [4] and can give rise to almost all lineages and are promising cells for cellular therapy [1]. From the very first derivation of human ESCs, scientists are keenly interested in the use of ESCs for drug discovery, immunotherapy and regenerative medicine, but their use has been restricted due to ethical issues and also because of difficulty in obtaining quality human oocytes.

Induced pluripotent stem cells (iPSCs) are generated from adult cells by the overexpression of four transcription factors Oct4/3 (octamer-binding transcription factor 4/3), Sox2 (sex determining region Y), Klf4 (kruppel-like factor 4) and c-Myc (Avian Myelocytomatosis virus oncogene cellular homologue) [5]. The iPSCs at cellular level are almost similar to ESCs as they are having the capacity of self-renewal, differentiation potential and the ability to produce germ line competent-chimeras. After these findings, two groups Takahashi et al. [6] and Nakagawa et al. [7] have generated the iPSCs from adult human fibroblasts. Though iPSCs possess great potential for cell therapy, but their genomic stability is still questionable.

Around the world, scientists are researching for stable, safe and highly accessible stem cells source with great potential for regenerative medicine. The cells isolated from mouse bone marrow upon culture exhibited the plastic adherence properties and formed spindle-shaped colonies were referred as colony forming unit fibroblasts [8]. Due to their ability to differentiate into specialized cells developing from mesoderm, they were named as mesenchymal stem cells (MSCs). MSCs, also known as multipotent cells, exist in adult tissues of different sources, ranging from murine to humans. They are self-renewable, multipotent, easily accessible and culturally expandable invitro with exceptional genomic stability and few ethical issues, marking its importance in cell therapy, regenerative medicine and tissue repairment [9].

The current review highlights recent findings in the areas of hMSCs (human MSCs) sources, its ex vivo differentiation ability, immunogenicity, homing ability, banking and cryopreservation, its role in the treatment of chronic diseases and its use in human clinical trials.

Since the first description of hMSCs derived from bone marrow [10], they have been isolated from almost all tissues including perivascular area [11]. Still there is neither a single definition nor a quantitative assay to help in the identification of MSCs in mixed population of cells [9]. However, the International Society for Cellular Therapy has proposed minimum criteria to define MSCs. These cells (a) should exhibits plastic adherence (b) possess specific set of cell surface markers, i.e. cluster of differentiation (CD)73, D90, CD105 and lack expression of CD14, CD34, CD45 and human leucocyte antigen-DR (HLA-DR) and (c) have the ability to differentiate invitro into adipocyte, chondrocyte and osteoblast [12]. These characteristics are valid for all MSCs, although few differences exist in MSCs isolated from various tissue origins.

MSCs are present not only in fetal tissues but also in many adult tissues with few exceptions. Efficient population of MSCs has been reported from bone marrow [10]. Cells which exhibits characteristics of MSCs were isolated from adipose tissue [13,14], amniotic fluid [15,16], amniotic membrane [17], dental tissues [18,19], endometrium [20], limb bud [21], menstrual blood [22], peripheral blood [23], placenta and fetal membrane [24], salivary gland [25], skin and foreskin [26,27], sub-amniotic umbilical cord lining membrane [28], synovial fluid [29] and Wharton's jelly [30,31] ().

Summary of hMSCs sources, cell surface markers and expansion media with serum supplements

There are different protocols reported previously in terms of isolation, characterization and expansion of MSCs, but all MSCs (despite of protocol) exhibits the minimum criteria proposed by International Society for Cellular Therapy.

hMSCs were isolated based on their ability to adhere to plastic surface, but this method resulted in the formation of heterogeneous cells (stem cells along with their progenitor cells) [32]. Bone marrow-derived MSCs (BM-MSCs) are considered the best cell source and taken as a standard for the comparison of MSCs from other sources.

Establishment of a comprehensive procedure for the isolation, characterization and expansion of MSCs is the key to success for the use of these cells as a good source for regenerative medicine [33]. Unlike bone marrow, MSCs from other tissues can be easily obtained by non-invasive methods and its proliferation can be maintained up to many passages [34,35]. MSCs from bone marrow, peripheral blood and synovial fluid were isolated by using Ficoll density gradient method with small modifications [24,30,36] and seeded into culture plates. While isolating MSCs from bone marrow, some haematopoietic cells also adhere to the plastic plate but during sub-culturing these cells are washed away, leaving only adherent fibroblast like cells [37]. MSCs from various tissue sources (adipose, dental, endometrium, foreskin, placenta, Wharton's Jelly) were isolated after digestion with collagenase and then cultured at varying densities [20,25,33]. Recently an efficient method to isolate BM-MSCs using novel marrow filter device is explored [38], which is less time consuming and avoids the risk of external contamination. MSCs isolated from different sources were cultured using condition media such as Dulbecco's modified Eagle's media (DMEM) [25,33], DMEM-F12 [17,20,26], MEM [19,23,29], DMEM-LG [21,24], DMED-HG [27,28] and RPMI (Roswell Park Memorial Institute medium) [39]. The primary culture media was supplemented with 10% FBS [25,33], new-born calf serum (NBCS) [23] or fetal calf serum (FCS) [25] (). Besides the culture media and supplementation, the oxygen concentration also affects the expansion and proliferation of MSCs [40]. MSCs expansion is also documented when cultured in DMEM with low glucose supplemented with growth factors like fibroblast growth factor (FGF), epidermal growth factor (EGF) and B27 [27]. But most commonly DMEM with 10% FBS is vastly employed in culturing and expanding MSCs invitro; however, the use of exogenous FBS is highly debated.

According to the International Society for Cellular Therapy standard criteria, expression of specific set of cell surface markers is one of the essential characteristics of hMSCs. Those cells which are positive for CD73, D90, CD105 whereas negative expression of CD14, CD34, CD45 and HLA-DR are considered as MSCs. However, the most characterized and promising markers with highest specificities for MSCs are describe in the present study (). MSCs have been reported from various human tissues, which exhibit the expression of above mentioned cell surface markers along with positive expression of CD29, CD44, CD146, CD140b specific to tissue origin. The expression of CD34, which is a negative marker, is still controversial [41]. A number of studies have also reported that stage-specific embryonic antigen (SSEA)-4 [13,42], CD146 [43,44] and stromal precursor antigen-1 (Stro-1) [45] are the stemnes markers for MSCs. The human amniotic fluid-derived MSCs exhibits the expression of CD29, CD44, CD90, CD105, HLA-ABC [major histocompatibility complex class I (MHC I)] along with SH2 (Src homology 2), SH3 (Src homology 3), SH4 (Src homology 4) but lack the expression of HLA-DR (MHC II) [16]. Stro-1, which is consider as stemnes marker for MSCs, is reported positive in dental [46] and bone marrow [47,48] whereas negative in human adipose-derived MSCs (AD-MSCs) [49].

Although MSCs have great advantages over other stem cells, their clinical applications are hindered by many research barriers. One of the major challenges is to obtain adequate number of cells as these cells were found to lose their potency during sub-culturing and at higher passages. One of the reasons behind the senescence and aging of MSCs during invitro expansion is the decrease in telomerase activity [50]. It has been reported that human BM-MSCs become senescent during long-term culture, manifested by decline in differentiation potential, shortening of the telomere length and morphological alterations [51]. Similar results are also reported when MSCs derived from bone marrow and adipose tissues were progressively cultured at higher passages. The actual age of the cells in culture is usually determined by population doublings (PDs) time and MSCs colonies derived from a single cell has shown up to 50 PDs in 10weeks [52], whereas others have reported 30 PDs in approximately 18weeks [51]. However, culturing MSCs for a long time resulted in an increase in the probability of malignant transformation [53] and also showed decline in their multipotency. Early MSCs have proved higher differentiation ability to chondrocytes, adipocytes and osteocytes; however, at higher passages and on long-term culture, this differentiation property declines [54]. There are two vital compounds which influence MSCs properties during invitro culturing, serum and growth factors, which are associated with malignant transformation of MSCs at higher passages [54]. In minimal media condition, MSCs culturing requires 10% heat-inactivated FCS, but in such culture conditions the MSCs retain some FCS proteins, which may evoke immunologic response invivo [55]. Expanding MSCs in serum-free culture media showed a gradual decrease in differentiation potential and telomerase activity, but cells were resistant to spontaneous transformation and could be expanded at higher passages without any chromosomal alteration [54]. However, due to variation in culture media and growth factors used, the comparison of data is difficult.

hMSCs have the capacity to differentiate into all the three lineages, i.e. ectoderm, mesoderm and endoderm, with various potency by employing suitable media and growth supplements which initiate lineage differentiation ().

Invitro differentiation potential of hMSCs

In addition to multipotency and expressions of cell surface markers, one of the determining properties of MSCs is to differentiate into mesodermal lineages. The invitro differentiation into adipocytes, osteocytes and chondrocytes, confirmed by production of oil droplet, formation of mineralized matrices and expression of typeII collagen respectively, has been evaluated by immunocytochemical, histochemical and PCR analysis [10,5658]. Differentiation of MSCs into adipocytes is induced by proper media supplementations, which activate transcription factors (genes) responsible for adipogenesis. For adipogenesis, MSCs were cultured in growth medium supplemented with dexamethasone, indomethacine, insulin and isobutyl methyl xanthine for 3weeks and the cells were analysed by accumulation of lipid droplets and expression of adipocytes-specific genes peroxisome proliferator-activated receptor (PPAR), adipocyte protein 2 (ap2) and lipoprotein lipase (LPL) genes [10,59]. Induction of adipogenesis is characterized by two phases: determination phase and terminal differentiation phase [60]. During determination phase, the cells committed towards pre-adipocytes show similar morphology to fibroblasts and cannot be distinguished from their MSCs precursors; however, at terminal phase the pre-adipocytes become mature adipocytes and formed lipid droplets and express adipocytes-specific proteins [59]. Overall, adipogenesis is an ordered process, involving multiple signalling cascades which are further discussed later in the present review.

The classical method to differentiate MSCs into osteocytes is by culturing the cells with ascorbic acid, -glyceralphosphate and dexamethasone for 3weeks in growth conditioned media. The osteogenic induction of MSCs initiated mineral aggregation and showed increase in alkaline phosphatase activity at final week of differentiation [10]. These mineralized nodules were found positive for Alizarin Red and von Kossa staining. The process of osteogenesis starts with assurance of osteoprogenitor which first differentiate into pre-osteocytes and then finally differentiate into mature osteoblasts [61]. One of the most important indicating factors for osteogenesis is the expression of runt-related transcription factor 2 (Runx2) [61]; however, other transcription factors like osteonectin, bone morphogenic protein 2 (BMP2) and extracellular signal molecules along with Runx2 expression, are involved in this process. In the whole process of bone formation, first osteoblasts synthesize the bone matrix and then help in bone remodelling and mineral deposition.

The differentiation of MSCs into mesenchymal lineage is known to be controlled by diverse transcription factors and signalling cascades. Many investigators have reported that a correlation exists between adipogenesis and osteogenesis [62,63]. It was reported that a converse relationship exists between adipogenesis and osteogenesis during culturing with different media supplements. [64]. Several signalling pathways such as Hedgehog [65,66], NEL-like protein 1 (NELL-1) [63] and catenin-dependent Wnt [67,68] are well manifested for pro-osteogenic and anti-adipogenic stimulations in MSCs, although there are various signalling cascades which demonstrate positive regulation of both adipo- and osteogenesis. Among them, one of the most familiar clinically-relevant molecule is BMP, which promotes MSCs differentiation and its osteogenic commitment [69,70] and also induce pro-adipogenic effects [71]. PPAR and Runx2 are the key transcription factors which control the adipogenic and osteogenic signalling cascades and the expression of one transcription factor counteracts expression of other transcription factor [14,72].

Like the adipogenesis and osteogenesis, hMSCs have the potential to differentiate into mature chondrocytes. The first standard protocol for chondrocytes differentiation was established for MSCs derived from human bone marrow [73]. According to the standard protocol for chondrogenesis, cells were cultured in DMEM media supplemented with insulin transferrin selenium, linoleic acid, selenious acid, pyruvate, ascorbate 2-phosphate, dexamethasone and transforming growth factor- III (TGF-III). The pre-induction stage of chondrogenic differentiation of MSCs resulted in the formation of pre-chondrocytes and expresses typeI and typeII collagens [74]. The expression of these genes and other adhesion molecules depends on the presence of soluble factors, i.e. TGF- family (TGF-1, TGF-2 and TGF-3) [75]. In the final step, pre-chondrocytes differentiate into mature chondrocytes and express chondrogenic transcription factors like Sox9, L-Sox5 and Sox6 [76,77]. In association with TGF-1, other growth factors such as, insulin like growth factor-I (IGF-I) and BMP-2 were known to induce the differentiation of MSCs into chondrocytes [78]. In hMSCs, TGF-1 interacts with Wnt/-catenin pathways inhibits osteoblast differentiation and induce chondrogenesis [79]. When human AD-MSCs were treated with BMP-2, they differentiated into chondrocytes and expressed mature cartilage markers (type II collagen/GAG) [80]. Besides these growth factors, other hormones such as parathyroid hormone-related peptide (PTHrp) [81,82] and triiodothyronine (T3) also influenced chondrogenesis.

Like cardiomyocytes, MSCs can differentiate into other mesodermal lineages. Twenty years ago, the rat BM-MSCs were cultured with 5-azacytidine which resulted in the differentiation of these cells into multinucleated myotubes [83]. Later Xu et al. [84] treated human BM-MSCs with the same chemical and demonstrated that the cells differentiate into myocytes and were expressing myocyte-related genes, -myocin heavy chain, -cardiac actin and desmin with additional calciumpotassium-induced calcium fluxes. Human BM-MSCs also differentiate into skeletal muscles and smooth muscles when transfected with notch intracellular domain (NICD) [85] followed by treatment with TGF- [86]. Yet the exact invivo signalling mechanism which initiates the differentiation of hMSCs into myocytes is not completely understood and under investigation.

Despite the mesodermal origin, hMSCs have displayed the capacity of trans-differentiation into ectodermal lineages. The hMSCs isolated from different sources have demonstrated trans-differentiation into neuronal cells upon exposure to neural induction media supplemented with cocktails of growth factors. Several growth factors like hepatocyte growth factor (HGF), FGF and EGF were used in neuronal induction media cocktail and successfully obtained neuronal specific phenotypes, i.e. oligodendrocytes, cholinergic and dopaminergic neurons [8791]. Barzilay et al. [89] reported that a transcription factor neurogenin-1 was found effective in the trans-differentiation of MSCs into neuronal protein expressing cells. In another study, a LIM homoeobox transcription factor 1 (LMX1a) expression into human BM-MSCs resulted in differentiation to dopaminergic neurons [89]. When BM-MSCs were cultured in serum-free media with forskolin and cAMP, cells attained neuronal morphology and elevated the expression of neuronal-specific markers [92]. -Mercaptoethanol (BME)- and nerve growth factor (NGF)-treated MSCs also differentiated into cholinergic neuronal cells [87]. Many studies have shown that factors like insulin, retinoic acid, bFGF, EGF, valproic acid, BME and hydrocortisone support neuronal differentiation of AD-MSCs [93,94]. Glial cell line-derived neurotrophic growth factors (GNDF), brain-derived neurotrophic factors (BDNF), retinoic acid, 5-azacytidine, isobutylmethylxanthine (IBMX) and indomethacin enhanced the MSCs differentiation into mature neuronal cells [95]. Gangliosides are glycosphingolipids which interact with EGF receptor (EGFR) and enhance osteoblast formation. However, reduction in gangliosides biosynthesis leads to inhibition of neuronal differentiation [96]. Human umbilical cord blood-derived MSCs (UCB-MSCs) co-transfected with telomerase reverse transcriptase (TERT) and BDNF revealed a longer life span and maintained neuronal differentiation which was effective in recovery of hypoxic ischaemic brain damage (HIBD) [97]. The dental derived MSCs, which originate from neural crest, successfully differentiated into mature neuronal cells [98,99]. hMSCs originate from mesoderm but have the potential to transdifferentiate into neural cells which can revolutionize the regenerative cell therapy in treating many neurological disorders.

It was believed that hepatocytes could only be derived from the cells originating from endoderm and their progenitor cells. However, MSCs have revealed the capacity of trans-differentiation into hepatocytes and pancreocytes upon induction with their corresponding conditioned media. Human BM-MSCs were trans-differentiated into hepatocyte by using two steps protocol: differentiation step followed by maturation step. In differentiation step, cells were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with EGF, bFGF and nicotinamide for a week. Finally during maturation step, differentiated human BM-MSCs were cultured with IMDM supplemented oncostatin M, dexamethasone and ITS+ (insulin, transferrin, selenium) premix which resulted in mature hepatocytes [100,101]. The hepatocyte-differentiated cells expressed liver-specific transcription markers, i.e. albumin, -fetoprotein, nuclear factor 4 (HNF-4); however, the differentiation capacity remains inadequate for clinical application. Among these transcription factors, HNF-4 is an essential transcription factor for the morphological and functional differentiation towards hepatocytes [102,103]. When human UCB-MSCs were transduced with HNF-4, it enhanced the differentiation capacity of the cells and increased expression of liver-specific markers [104]. In other studies, it was shown that valproic acid, which is histone deacetylase inhibitor, up-regulate the expression of hepatic marker through activation of protein kinase B (AKT) and extracellular signal-regulated kinases (ERK) [105].

Human BM-MSCs have been successfully differentiated into insulin producing -cells invitro and transplanted to streptozotocin-induced diabetic mice which corrected the hyperglycaemic condition [106,107]. The paracrine factors increase the differentiation and maturation of human BM-MSCs into pancreatic lineage without any genetic manipulation [108]. Human dental pulp stem cells also differentiated into insulin producing cells by induction with growth factors, i.e. acitvin A, sodium butyrate, taurine and nicotinamide [109]. Till now hMSCs derived from adipose, dental, umbilical cord, amnion, Wharton jelly and placental tissues have successfully differentiated into insulin producing -cells [110112]. These studies have revealed that hMSCs can differentiate into endodermal lineages which can transform the current traditional drug therapies to a future promising cell based therapies.

Regarding clinical research on cellular therapy, it is very important to know about the immunomodulatory capabilities of MSCs. In the current era of cell therapy and transplantation, the infusion of MSCs and host compatibility is the main subject of interest. Due to low expression of MHC I and lack expression of MHC class II along with co-stimulatory molecules, like CD80, CD40 and CD86, MSCs are unable to bring substantial alloreactivity and these features protects MSCs from natural killer (NK) cells lysis [113]. The MSCs therapy might alleviate disease response by increasing the conversion from Th2 (T helper cells) response to Th1 cellular immune response through modulation of interleukin (IL)-4 and interferon (IFN)- levels in effector T-cells [114]. MSCs have the ability to inhibit the NK cells and cytotoxic T-cells by means of different pathways. The secretion of human leucocytes antigen G5 was also found helpful in the suppression of T lymphocytes and NK cells [115]. By the secretion of suppressors of T-cells development [116], inhibitory factors i.e. leukaemia inhibitory factor (LIF) [117] and IFN- [118] enhance immunomodulatory properties of MSCs. Moreover, it is observed that human BM-MSCs were not recognized by NK cells, as they expressed HLA-DR molecules [119]. When allogenic hMSCs were transplanted into patients, there was no production of anti-allogeneic antibody nor T-cell priming [120], but the cytotoxic immune factors were found to be involved in the lysis of MSCs [114,121]. In this situation, the IFN- act as antagonist of NK cells, i.e. IL-2-treated NKs are recognized to destroy MSCs whereas IFN- helps the MSCs to keep it safe from NKs [122]. In the same report, Jewett et al. [122] mentioned that along with the protection of MSCs from cytotoxic factors, IFN- also enhances the differentiation of these cells by nuclear factor kappa (NFB)-dependent and -independent pathway. Toll-like receptors (TLRs) are the key components of innate immune system, which is critically involved in the initiation of adaptive immune system responses. MSCs have the expression of TLRs that elevate their cytokines secretions as well as proliferation [123]. MHC class I chain-like gene A (MICA) together with TLR3 ligand and other immunoregulatory proteins kept the MSCs safe from NKs invasion [123]. Together with other properties, these immunomodulatory features makes MSCs one of the feasible stem-cells source for performing cell transplantation experiments.

Considering the homing ability, multilineage potential, secretion of anti-inflammatory molecules and immunoregulatory effects, MSCs are considered as promising cell source for treatment of autoimmune, inflammatory and degenerative diseases. Efforts have been made to discuss the role of MSCs in treating chronic diseases in animal disease model ().

hMSCs and chronic diseases

We previously discussed that MSCs have the ability to differentiate into neurons [8799]. The first MSCs transplantation for neurodegenerative disorder was conducted in acid sphingomyelinase mouse model. After the injection of MSCs, there was a decrease in disease abnormalities and improvement in the overall survivability of the mouse [124]. Based on this experiment, a new study was designed to ascertain the potency of MSC transplantation into amylotrophic lateral sclerosis (ALS), a neurodegenerative disease that particularly degenerate the motor neurons and disturb muscle functionality [124]. The MSCs were isolated from the bone marrow of patients and then injected into the spinal cord of the same patients, followed by tracking of MSCs using MRI at 3 and 6 months. As a result, neither structural changes in the spinal cord nor abnormal cells proliferation was observed. However, the patients were suffering from mild adverse effects, i.e. intercostal pain irradiation and leg sensory dysesthesia which were reversed in few weeks duration. In another study, the AD-MSCs were genetically modified to express GDNF and then transplanted in rat model of ALS which improved the pathological phenotype and increased the number of neuromuscular connections [125].

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by substantial loss of dopaminergic neurons. The MSCs enhanced tyrosine hydroxylase level after transplantation in PD mice model [126]. MSCs by secretion of trophic factors like vascular endothelial growth factor (VEGF), FGF-2, EGF, neurotrophin-3 (NT3), HGF and BDNF contribute to neuroprotection without differentiating into neurocytes [127,128]. Now new strategies are being adopted like genetic modifications of hMSCs, which induce the secretions of specific factors or increase the dopamine (DA) cell differentiation. BM-MSCs were transduced with lentivirus carrying LMX1a gene and the resulted cells were similar to mesodiencephalic neurons with high DA cell differentiation [89]. Research group from the university hospital of Tubingen in Germany first time delivered MSCs through nose to treat neurodegenerative patients. The experiments were performed on Parkinson diseased rat with nasal administration of BM-MSCs [129]. After 4.5 months of administration, MSCs were found in different brain regions like hippocampus, cerebral, brain stem, olfactory lobe and cortex, suggesting that MSCs could survive and proliferate invivo successfully [129]. Additionally, it was observed that this typeof administration increased the level of tyrosine hydroxylase and decreased the toxin 6-hydroxydopamine in the lesions of ipsilateral striatum and substantia nigra. This novel delivery method of MSCs administration could change the face of MSCs transplantation in future.

Alzheimer disease (AD) is one of the most common neurodegenerative disease. Its common symptoms are dementia, memory loss and intellectual disabilities. Till now no treatment has been established to stop or slow down the progression of AD [130]. Recently, researchers are in the search to reduce the neuropathological deficits by using stem cell therapy in AD animal model. It was demonstrated that human AD-MSCs modulate the inflammatory environment, particularly by activating the alternate microglia which increases the expression of A-degradation enzymes and decreases the expression of pro-inflammatory cytokines [131]. Furthermore, it was observed that MSCs modulate the inflammatory environment of AD and inadequacy of regulatory T-cells (Tregs) [132] and later on it was reported that they could modulate microglia activation [133]. It was previously demonstrated that human UCB-MSCs activate Tregs which in turn regulated microglia activation and increased the neuronal survival in AD mice model [134]. Most recently, it was evidenced that MSCs enhanced the cell autophagy pathway, causing to clear the amyloid plaque and increased the neuronal survivability both invitro and invivo [135].

MSCs are also used to assuage immune disorders because MSCs have the capacity of regulating immune responses [1]. After revealing the facts that human BM-MSCs could protect the haematopoietic precursor from inflammatory damage [136], other hMSCs can be used for the treatment of autoimmune diseases.

Rheumatoid arthritis (RA) is a joint inflammatory disease which is caused due to loss of immunological self-tolerance. In preclinical studies on animal models, MSCs were found helpful in the disease recovery and decreasing the disease progression. The injections of human AD-MSCs into DBA/1 mice model resulted in the elevation of inflammatory response in the animal [137]. They further demonstrated that following the injections of AD-MSCs, the Th1/Th17 antigen-specific cells expansion took place due to which the levels of inflammatory chemokines and cytokines reduced, whereas this treatment increased the secretion of IL-10 [138]. Along with its anti-inflammatory function, IL-10 is an important factor in the activation of Tregs that controls self-reactive T-cells and motivates peripheral tolerance invivo [138]. Similar to this, human BM-MSCs demonstrated the same results in the collagen-induced arthritis model in DBA/1 mice [139]. These studies suggest that MSCs can improve the RA pathogenesis in DBA/1 mice model by activating Treg cells and suppressing the production of inflammatory cytokines. However, some contradictions were reported in adjuvant-induced and spontaneous arthritis model, showing that MSCs were only effective if administered at the onset of disease, which suggests that on exposing to inflammatory microenvironment MSCs lost their immunoregulatory properties [140].

Type 1 diabetes is an autoimmune disease caused by the destruction of -cells due the production of auto antibody directed against these cells. As a result, the quantity of insulin production reduces to a level which is not sufficient to control the blood insulin. It has been demonstrated that MSCs can differentiate into insulin producing cells and have the capacity to regulate the immunomodulatory effects [118]. For the first time, nestin positive cells were isolated from rat pancreatic islets and differentiated into pancreatic endocrine cells [141]. Nestin positive cells were isolated from human pancreas and transplanted to diabetic nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice, which helped in the improvement of hyperglycaemic condition [142]. However, these studies were found controversial and it was suggested that besides pancreatic tissues, other tissues can be used as an alternative for MSCs isolation to treat type1 diabetes. Human BM-MSCs were found effective in differentiating into glucose competent pancreatic endocrine cells invitro as well as invivo [108]. Studies on UCB-MSCs presented a fascinating option for the use of these cells for insulin producing cells. It was demonstrated that UCB-MSCs behave like human ESCs, following similar steps to form the differentiated -cells [143]. The most recent findings of Unsal et al. [144] showed that MSCs when transplanted together with islets cells into streptozotocin treated diabetic rat model enhance the survival rate of engrafted islets and are found beneficial for treating non-insulin-dependent patients in type1 diabetes.

For myocardial repair, cardiac cells transplantation is a new strategy which is now applied in animal models. MSCs are considered as good source for cardiomyocytes differentiation. However, invivo occurrence of cardiomyocytes differentiation is very rare and invitro differentiation is found effective only from young cell sources [145,146]. MSCs trans-differentiated into cardiomyocytes with cocktail of growth factors [84] were used to treat myocardial infarction and heart failure secondary to left ventricular injury [147]. The systematic injection of BM-MSCs into diseased rodent models partially recompensed the infarcted myocardium [148,149]. Furthermore Katrisis et al. [150] transplanted autologous MSCs along with endothelial progenitor cells and evidenced the improvement in myocardial contractibility, but they did not decrypt the mechanism which brought out these changes. Although MSCs are effective in myocardial infarction and related problems, but still cell retentivity in the heart is rapidly decreased, after 4h of cells injection only 10% and after 24h it was found approximately 1% cell retention [151,152]. Following this study, Roura et al. [153] reported that UCB-MSCs retained for several weeks in acute myocardial infarction mice, proliferated early and then differentiated into endothelial lineage. Most recently, transplantation of UCB-MSCs into myocardial infarction animal model along with fibronectin-immobilized polycaprolactone nanofibres were found very effective [154]. All these studies collectively indicate the role of hMSCs in cellular therapy of cardiac infarction and currently there are approximately 70 registered trials investigating the effect of MSCs therapy for cardiac diseases (clinicaltrials.gov).

Homing is the term used when cells are delivered to the site of injury, which is still challenging for cell-based therapies. Most of the time local delivery and homing of cells are found beneficial due to interaction with the host tissues, accompanied by the secretion of trophic factors [114]. There are a number of factors, like cells age, culturing conditions, cell passage number and the delivery method, which influence the homing ability of MSCs to the injured site.

Higher passage number decreases the engraftment efficiency of MSCs and it has been shown that freshly isolated MSCs had greater homing efficiency than the cultured cells. Besides this, the source from which MSCs are being isolated also influences the homing capacity of MSCs. While culturing MSCs, it was shown that oxygen condition, availability of cytokines and growth factors supplements in the culture media triggers important factors which are helpful in the homing of MSCs. Matrix metallo-proteases (MMPs), the important proteases which are involve in the cell migration, also plays important role in the MSCs migration [155]. The higher cell numbers and hypoxic condition of the culturing environment influence the expression of these MMPs [156]. The inflammatory cytokines, i.e. IL-1, TNF- and TGF-1 , enhance the migration of MSCs by up-regulating the level of MMPs [155]. The next important factor is delivery method via which the MSCs are administered to the desired tissue. Intravenous infusion was the most commonly administered route [157], because if MSCs were administered systemically it will trap in the capillaries sheet of various tissues, especially in lungs [158]. That is the reason why most of the time intra-arterial injections of MSCs has been advised, but the most convenient and feasible way of MSCs transplantation is local injection to the site of injury or near the site of injury which provides more number of cells and increases its functional capacity.

The exact mechanism via which MSCs migrate and home to the injured site is still unknown, although it is believed that certain chemokine and its receptors are involved in the migration and homing of MSCs to the tissue of interest. MSCs express many receptors and adhesion molecules which assist in its migration process. The chemokine receptor type4 (CXCR4) and its binding protein stromal-derived factor 1- (SDF-1) play a vital role in this process [159]. In order to know the homing capacity and to monitor the therapeutic efficiency of MSCs, invivo tracking by non-invasive method are pre-requisite. Some advance techniques, i.e. single photon emission CT (SPECT), bioluminescence imaging (BLI), positron emission tomography (PET) were being applied for tracking the MSCs.

As we discussed earlier that MSCs have higher trans-differentiation potential and exhibits immunomodulatory features, but their off target homing, especially lodging in the lungs, is a major obstacle. There is need for in-depth study of MSCs homing mechanism and finding appropriate tracking without any negative effect on the cells and host.

From all the previous studies, it is obvious that the use of hMSCs for clinical applications will increase in future. For clinical applications, a large number of MSCs in an off the shelf format are required. For this purpose, a proper set up of invitro MSCs expansion and subsequent cryopreservation and banking are necessary to be established. This will provide unique opportunities to bring forward the potential uses and widespread implementation of these cells in research and clinical applications. Keeping in mind its use in future clinical and therapeutic applications, there is a need to ensure the safety and efficacy of these cells while cryopreserving and banking. For the selection of optimal cryopreservation media, uniform change in temperature during freezing and thawing, employed freezing device and long-term storage in liquid nitrogen are the indispensable factors to consider.

First considerable factor is the optimal cryopreservation media in which cells can maintain their stem cells abilities for long time. In the cryopreservation media, the cells require the animal base reagent, like FBS, as a source of their nutrients, but previous studies have suggested that animal proteins are difficult to remove from the hMSCs and that these resident protein may enhance adverse reactions in the patients who receive these cells for treatment [35]. Therefore, a serum-free media is substantial for the cryopreservation of MSCs and researchers have successfully used the serum-free media for cryopreservation of MSCs [160,161]. Most recently, human albumin and neuropeptide were used instead of FBS and MSCs maintained their cell survival and proliferation potential in the culture conditions. Additionally, cryoprotective agents (CPAs) are required for the cryopreservation media to prevent any freezing damage to cells. A large number of CPAs are available [162] among which DMSO is the most common CPAs used in cryopreservation of MSCs. However, DMSO is toxic to both humans and animals which make it complicated in the use of MSCs freezing for clinical applications and it has been showed that DMSO has bad effects in both animals and humans [163]. On the infusion of MSCs frozen in DMSO, patients develop mild complications like nausea, vomiting, headache, hypertension, diarrhoea and hypotension [164] and also severe effects like cardiovascular and respiratory issues were reported [165]. Due to these toxic effects, it is necessary to remove (washing with isotonic solutions) or replace DMSO with an alternate CPA. There are several methods along with the introduction of automated cells washing for the removal of DMSO from the frozen thawed cells [166]. Most recently for tissue cryopreservation, a new method was introduced using the mixture of 0.05M glucose, 0.05M sucrose and 1.5M ethylene glycol in phosphate buffer saline [167], shown successful isolation and characterization of MSCs after 3 months of cryopreservation of the tissue. Hence, this method is without any DMSO and animal serum, but it is not yet applied for MSCs cryopreservation. From these findings, it is clear that for clinical grade cells, there is a need of a cryopreservation protocol either with low concentration of DMSO or to replace DMSO with non-toxic alternative.

For cryopreservation of MSCs, the second important factor is the freezing temperature rate. Mostly slow freezing at the rate of 1C/min is the optimum rate for MSCs preservation [168]. For this purpose, current controlled rate freezers (CRFs) are suitable for controlling temperature, maintaining the rate of temperature during cryopreservation. These CRFs can be programmed to find out the exact temperature which the sample is experiencing during freezing [169]. Despite of these benefits, these CRFs lack the uniformity of temperature to all vials during large-scale banking of MSCs [170], so for large-scale banking, the development of advance CRFs are mandatory. Recently more advanced CRF, which provides unidirectional flow of cryogen to each sample, were created by Praxair Inc. On large-scale MSCs banking, along with the safe and efficient cryopreservation, the regulatory guidelines are also important. Like in the U.S.A., Food and Drug Administration (FDA) is responsible whereas in Europe, European Medicines Agency is responsible in Europe for supervising MSCs based cell therapy products.

MSCs have a promising future in the world of clinical medicine and the number of clinical trials has been rising since the last decade. Along with preclinical studies, MSCs have been found to be persuasive in the treatment of many diseases [1]. A large number of clinical trials have been conducted and this trend is gradually increasing (). Currently, there are 463 registered clinical trials in different clinical phases (phase I, II etc.), evaluating the potential of MSC-based cell therapy throughout the world (ClinicalTrials.gov). Most of these trials are phase I/II studies and combination of phase II/III studies, whereas very small numbers of these trials are in phase IV or phase III/IV. Among 463 registered trials, 264 trials are in open status which is open for recruitment whereas 199 trials are closed; out of which 106 studies are completed whereas the rest are in active phases. Clinical trials conducted with MSCs showed very less detrimental effects; however, few of them showed mild adverse effects. Due to immunomodulatory properties, MSCs have been used in many human autoimmune disease clinical trials. However, the exact mechanism by which MSCs regulate the immune response is unclear [171]. To date, 45 autoimmune-disease clinical trials have been registered, out of which seven are completed, 22 are open for recruitment whereas the rest are in active phases (ClinicalTrials.gov). Similarly 70 trials are registered for cardiovascular diseases, 37 for osteoarthritis, 32 for liver disorders, 29 for graft versus host disease (GvHD), 21 for respiratory disorders, 15 for spinal cord injury, 15 for kidney failure, 13 for skin diseases, seven for muscular dystrophy, five for aplastic anaemia, four for Osteogenesis imperfecta, four for AD, two for PD, two for ulcerative colitis and rest are for other diseases (). Although the progress of clinical studies so far registered is slow (only seven studies with final results), but the efficient use of MSCs in large clinical trials with upcoming promising results have proven MSCs as boon for regenerative medicine.

Number of clinical trials registered (per year) for MSCs based therapy (ClinicalTrials.gov)

Number of common diseases registered for MSCs based cell therapy (ClinicalTrials.gov)

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Mesenchymal Stromal Cells – PubMed Central (PMC)

Curr Opin Hematol. Author manuscript; available in PMC 2012 Jun 1.

Published in final edited form as:

PMCID: PMC3365862

NIHMSID: NIHMS16948

Mesenchymal stromal cells (MSCs) are the spindle shaped plastic-adherent cells isolated from bone marrow, adipose, and other tissue sources, with multipotent differentiation capacity in vitro. However, whether MSCs truly qualify as stem cells is an area of some debate[1]. MSCs were first described by Friendenstein as hematopoietic supportive cells of bone marrow. He showed that MSCs could differentiate to bone in vitro and a subset of the cells had a high proliferative potential (CFU-F) when plated at low density in tissue culture[2,3]. Based largely on Friendensteins work, Maureen Owen proposed the existence of a stromal stem cell to maintain the marrow microenvironment as the hematopoietic stem cell maintains hematopoiesis[4]. The notion of a mesenchymal stem cell was popularized by Arnold Caplan proposing that MSCs gave rise to bone, cartilage, tendon, ligament, marrow stroma, adipocytes, dermis, muscle and connective tissue[5]. However, convincing data to support the stemness of these cells were not forthcoming, and now most investigators recognize that in vitro isolated MSCs are not a homogenous population of stem cells, although a bona fide mesenchymal stem cell may reside within the adherent cell compartment of marrow[6].

MSCs undoubtedly play a critical role in the marrow microenvironment. Following intramedullary transplantation of eGFP-marked human MSCs into a NOD SCID mouse, the MSCs incorporated into the murine marrow microenvironment and improved the human hematopoietic stem cell activity in the host mouse[7]. MSCs are also thought to be of great value for cell based therapies. This discussion will focus on the properties of MSCs that engender their utility as therapeutic cells and specifically on MSCs as treatment for GVHD and as targeting vehicles for anti-tumor therapies.

As stated above, data to support the designation of MSCs as biologically functional stem cells are lacking. However, the acronym, MSC, is firmly engrained in the vernacular of cell biologists and clinical cell therapists. Thus, the International Society for Cellular Therapy (ISCT) has recommended that these spindle-shaped, plastic-adherent cells be termed, mesenchymal stromal cells [6]. This label allows investigators to continue to use the acronym, MSCs, which should reduce the potential for confusion in the literature. A biologically active stem cell for mesenchymal tissues may exist, but the term mesenchymal stem cell should be reserved for the subset of mesenchymal cells that demonstrate stem cell activity by rigorous criteria.

The defining characteristics of MSCs are inconsistent among investigators due, in part, to the lack of a universally accepted surface marker phenotype. However, all proposed MSC populations are plastic adherent in vitro; hence, this is one defining characteristic. The first important studies of surface antigen markers led to the development of SH2 and SH3, antibodies which seemed to identify MSCs[8]. Subsequently, SH2 and SH3 were shown to recognize epitopes on CD105 and CD73, respectively[9,10]. Furthermore, CD90 is expressed on all cells that we accept as MSCs. These cells do not express hematopoietic antigens, e.g. CD45, CD34, CD14, CD19, or CD3. Additionally, MSCs express MHC Class I molecules in vitro, but not Class II molecules unless stimulated, e.g. by interferon, in tissue culture. Thus, a surface marker phenotype of MSCs is CD105+, CD73+, CD90+, CD45, CD34 CD14, CD19, CD3, HLA DR. While unequivocally identifying MSCs, this surface marker profile is cumbersome. Stable, pancellular expression of surface markers that are unique to MSCs within the bone marrow, the most common source of MSCs, would greatly facilitate the identification of these cells.

The single most characteristic feature of MSCs is the capacity to differentiate to osteoblasts, adipocytes, and chondroblasts in vitro. It is therefore quite reasonable for investigators to demonstrate such trilineage differentiation in vitro to prove their cells under study are MSCs.

In practice, MSCs can be defined by the criteria shown in the Table, as proposed by the ISCT Mesenchymal and Tissue Stem Cell Committee[11]. The criteria are designed not only to define the MSCs, but also to exclude hematopoietic cells, which is important since, as stated above, MSCs are most commonly isolated from bone marrow. CD3 expression is not included in the criteria because T cells are uncommon contaminants of MSC preparations. It is important to avoid hematopoietic cells among the populations of MSCs being used for cell therapy studies because they could alter the scientific outcomes and may be deleterious for patients in clinical trials.

For obvious reasons, if the proposed therapeutic cells are not readily accessible, clinical utility is limited. Effective cell therapy, therefore, begins with a cell type that is relatively easy to isolate. MSCs are most often isolated by adherence selection. For example, bone marrow mononuclear cells are placed in a plastic tissue culture vessel and maintained for 15 days at 37C. Then, the nonadherent cells are removed as the media is changed and the remaining adherent cells are isolated MSCs. At this stage, the MSC cultures are definitely not free of contamination by resident tissue cells, e.g. hematopoietic cells; however, successive passages of the ex vivo expanded cells effectively remove most or all contaminating cells. Thus, tissue culture serves to expand and purify the MSCs. Similarly, when other sources of MSCs, e.g. adipose tissue, a mononuclear cell preparation is maintained in tissue culture to isolate the MSCs.

There are three fundamental questions that must be addressed when using MSCs as cell therapy for tissue regeneration. First, will MSCs differentiate to the tissue of interest in vivo? This is a critically important issue as certain culture conditions may induce atypical differentiation in vitro that may not occur in vivo. Additionally, MSCs may not differentiate to the targeted tissue, but instead generate cell types that function in a beneficial way within the tissue. For example MSCs may secrete useful soluble mediators that foster repair of a tissue so that differentiation is unneeded for clinical benefit. Thus, MSCs may be highly effective for applications in regenerative medicine by several mechanisms.

Second, how can the cells be delivered to the relevant tissue(s)? For example, if intravenously infused, will MSCs home to the desired sites? Although some investigators have suggested that MSCs home to sites of inflammation, it is unclear that MSCs home to sites of other types of local or systemic disease, and there is little data indicating that MSCs home to healthy tissue. Despite the uncertainty of homing to diseased tissues, sufficient intravenously infused MSCs may arrive and incorporate in the desired tissue to generate clinical benefits. For example, Horwitz et al. reported the infusion of MSCs after BMT into children with osteogenesis imperfecta, a metabolic bone disorder. Engraftment and growth acceleration was demonstrated in 5 of 6 patients[12]. Koc et al. reported MSC infusion in children with metachromatic leukodystrophy and Hurlers disease after BMT. In 4 of 6 patients with metachromatic leukodystrophy, an improvement in nerve conduction velocity was observed, but engraftment in the neural tissue was not assessed[13]. In both cases, homing strictly defined was not demonstrated; however the former study showed the presence of intravenously infused cells within the targeted tissue.

Third, how much tissue replacement by donor cells (i.e. engraftment) is needed to achieve correction or improvement of the damage or diseased tissue? The answer will likely be tissue and disease specific, and therefore will require animal models that reliably model the human disease, or more effectively, pilot clinical trials. Importantly, the level of tissue replacement is often quite low, far less than what may be hypothesized; consequently, estimates are useful to determine which diseases should be investigated, but experimental data are essential to formulate therapeutic strategies.

Any cell employed for therapeutic purposes would ideally be immunoprivileged allowing for use in HLA mismatched patients. Further, cells that can regulate the immune response could be effectively used to modulate the immune system to treat immunologic disease. MSCs have been reported to be immunosuppressive and immunoprivileged. The two terms are often used interchangeably; however, this is strictly incorrect. A cell may escape immune recognition (i.e. immunoprivileged) without having an effect on immune effector cells. Similarly, a cell may secrete immunosuppressive molecules while being recognized by an allogeneic immune system. MSCs do seem to exhibit an effect on the immune effector cells in vitro. This property has led to much dialogue whether MSCs could be effective therapy for autoimmune diseases such as rheumatoid arthritis. More important for this discussion is the role of MSCs in the treatment Graft-versus-Host Disease (GVHD).

As mentioned above, MSCs are an essential component of the stromal scaffold of the bone marrow that provides physical and functional support during hematopoiesis. Based on this concept, MSCs have been studied for their ability to improve engraftment of hematopoietic stem cells in vivo[14, 15]. While some reports suggest that MSCs increase engraftment, the data are not particularly impressive, at least in the models utilized. It has been recently shown that MSCs exert a profound immunomodulatory effect by means of both soluble and cell contact-dependent mechanisms[16]. MSCs can act both on T and B cells and although several mechanisms of action have been suggested, the data are contradictory. The ability to inhibit or stimulate T-cell alloresponses appears to be independent of HLA matching. It is still unclear whether MSCs naturally exhibit an immunoregulatory role or whether this is the consequence of a more general, non-specific interference with the cell cycle[17].

In this context, it is interesting to note that stromal cells, together with osteoblasts and endothelial cells, contribute to the formation of the HSC niche. This can be defined as a specialized microenvironment that precisely maintains a long-term storage of quiescent, slowly dividing HSCs by preventing their proliferation, differentiation or apoptosis. It can be hypothesized that MSCs, on one hand, are preventing T lymphocyte activation and proliferation (to prevent possible harm on HSC) and, on the other hand, seem to exert a potent anti-apoptotic effect. Although the mechanisms of immunomodulation are still unfolding, a relevant in vivo immunomodulatory effect has been shown: 1) if given in patients with severe acute GVHD, they are able to reverse the evolution of GVHD in a significant proportion of patients[18, 19], and 2) in a recent in vivo experiment in which injection of MSCs ameliorated the course of chronic progressive experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis[20].

The EBMT MSC Expansion Consortium used MSCs to treat grades IIIIV GVHD in 40 patients who were resistant to second line GVHD treatment. The MSC dose was a median 1.0 x 106 cells/kg recipient body weight (range 0.49 x 106 cells/kg). Adverse effects were not seen after MSC infusions. Nineteen patients received one dose, 19 patients received 2 doses, one patient received 3 doses, and one patient received 5 doses. In some cases, an individual patient received MSC doses from different donors. The MSC donors were HLA-identical siblings in 5 cases, haploidentical donors in 19 cases, and 41 cases of third-party HLA-mismatched donors. Among the 40 patients treated for severe acute GVHD, 19 had complete responses, 9 showed improvement, 7 did not respond, 4 had stable disease and 1 was not evaluated due to short follow-up. Ectopic tissue formation was not seen. MSC dramatically affected tissue repair of severe acute GVHD of the gut, liver, and skin in a consistent proportion of patients. Twenty-one patients are alive with between 6 weeks and 3.5 years follow-up after transplantation. Nine of these patients have extensive chronic GVHD. One patient with ALL has recurrent leukemia and one patient has de novo AML of host origin. In view of the dismal outcome in patients with grades IIIIV acute GVHD, the data from this small trial are promising. However, the optimal strategy for the treatment of GVHD based on MSC infusion has not yet been determined and remains rather complex for a several reasons: 1) the ex vivo cell expansion is expensive and time consuming; 2) there is variation in the expansion capability from donor to donor; 3) often, previously expanded MSCs are required for the timely treatment of GVHD; 4) the optimal dose of MSCs, or the need for multiple infusions, to obtain the maximal effect on GVHD is unknown; 5) expanded MSCs are very difficult to detect after infusion, and the patients marrow stroma remain of host origin with the possible exception of some pediatric patients.

Ongoing efforts within the EBMT Consortium are addressing these challenges in an effort to determine the role of MSC therapy in the treatment for GVHD. At the current state of research, we conclude that MSCs have both immunomodulatory and tissue repairing effects and should be further explored as treatment of severe acute GVHD in prospective randomized trials.

The formation of stroma is essential for tumor growth and involves complex interactions between malignant tumor cells and non-tumor stromal cells. Studeny et al. have demonstrated that MSCs integrate into solid tumors, suggesting the development of anti-cancer therapies based on the intratumoral production of agents by gene-modified MSCs[2123].

Andreeff and colleagues have now conducted a series of experiments to address this issue by noninvasively visualizing MSCs using luciferase bioluminescence. The cells were labeled by a fiber modified adenoviral vector expressing firefly luciferase (AdLux-F/RGD) and the MSC-Lux were injected into normal (healthy) SCID mice or mice bearing established metastatic breast or ovarian tumors. Biodistributed MSC-Lux were imaged utilizing the Xenogen IVIS detection system. In normal mice, human MSC (hMSC) migrated to the lungs where they remained resident for 710 days. In animals bearing established metastatic lung tumors, IV injected hMSC again migrated to the lungs. However, in contrast to control mice, the Lux signal remained strong over a 15-day period with only a slight decrease over the first 10 days. After IP injection, hMSC-LUX were detected in the peritoneum, and after 7 days, no hMSC-LUX was detected in normal animals, while strong punctate regions of LUX-activity were observed in ovarian tumors. In contrast to SCID mice injected with hMSC, when healthy Balb/C mice were injected, Balb/C derived MSC-LUX initially migrated to the lungs, but within 2.5 hrs had exited the lungs to remain liver and spleen resident for 57 days. Tumor cells were then transduced with renilla luciferase constructs allowing for the co-localization and dynamic interactions of firefly luciferase MSCs and renilla luciferase tumors to be demonstrated.

hMSC-producing interferon-beta (IFNb-MSC) were found to inhibit the growth of metastatic tumors in the lungs of SCID mice. When injected IV (4 doses of 106 MSC/week) into SCID mice bearing pulmonary metastases of carcinomas or melanomas, tumor growth was significantly inhibited as compared to untreated or vector-control MSC controls (p= 0.007), while recombinant IFNb protein (50,000 IU qod) was ineffective (p=0.14). IV injected IFNb-MSC prolonged the survival of mice bearing metastatic breast carcinomas (p=0.001). Intraperitoneal (IP) injections of IFN-MSC into mice carrying ovarian carcinomas resulted in doubling of survival in SKOV-3, and cures in 70% of mice carrying OVAR-3 tumors.

A similar strategy is also effective as therapy for brain tumors. MSC injected into the ipsilateral or contralateral carotid artery were found to localize to glioma xenografts in mice and IFNb-MSC significantly (p<0.05) prolonged survival of these mice[24].

These data suggest that systemically administered gene-modified MSC selectively engrafts into the tumor microenvironment and remain resident as part of the tumor architecture. MSC-expressing IFN-b inhibit the growth of melanomas, gliomas, metastatic breast and ovarian cancers in vivo and prolong the survival of mice bearing established tumors. Thus, MSCs are potentially a universal vehicle to deliver localized antitumor therapy. Clinical trials, which are in development, will be conducted to test these experimental findings.

MSCs have an enormous potential as cell therapy in tissue regeneration, immune modulation, and as delivery vehicles for the specific delivery vehicles for anti-tumor agents, but the true clinical utility remains to be proven. MSCs are relatively easy to isolate and purify, and we currently have means to unequivocally identify the cells, although more specific surface markers are needed. MSCs have been infused into well over a hundred patients, including young children, without serious adverse events testifying to the general safety of this strategy. Future efforts in our field must focus on better defining the therapeutic potential of MSCs through clinical trials and better understanding of the biology of MSCs to elucidate the mechanisms of these therapeutic effects.

Table. Summary of Criteria to Identify Mesenchymal Stromal Cells (MSCs).

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At 7% CAGR, Mesenchymal Stem Cells Market Size, Growth Set …

Apr 09, 2020 Xherald --Market Study Report Has Added A New Report On Mesenchymal Stem Cells Market That Provides A Comprehensive Review Of This Industry With Respect To The Driving Forces Influencing The Market Size. Comprising The Current And Future Trends Defining The Dynamics Of This Industry Vertical, This Report Also Incorporates The Regional Landscape Of Mesenchymal Stem Cells Market In Tandem With Its Competitive Terrain.

The global mesenchymal stem cells market size to reach USD 2,518.5 Million by 2026, growing at a CAGR of 7.0% during forecast period, according to a new research report published by The marker research report.

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The report 'Mesenchymal Stem Cells Market, [By Source (Bone Marrow, Umbilical Cord Blood, Peripheral Blood, Lung Tissue, Synovial Tissues, Amniotic Fluids, Adipose Tissues); By Application (Injuries, Drug Discovery, Cardiovascular Infraction, Others); By Region]: Market Size & Forecast, 2018 - 2026' provides an extensive analysis of present market dynamics and predicted future trends. The market was valued at USD 1,335.1 million in 2017. In 2017, the drug discovery application dominated the market, in terms of revenue. North America region is observed to be the leading contributor in the global market revenue in 2017.

are adult stem cells, which are traditionally found in the bone marrow. However, they can also be parted from other available tissues including peripheral blood, cord blood, fallopian tube. These stem cells mainly function for the replacement of damaged cell and tissues. The potential of these cell is to heal the damaged tissue with no pain to the individual. Scientists are majorly focusing on developing new and innovative treatment options for the various chronic diseases like cancer. Additionally, the local governments have also taken various steps for promoting the use of these stem cells.

The significant aspects that are increasing the development in market for mesenchymal stem cells consist of enhancing need for these stem cells as an efficient therapy option for knee replacement. Raising senior populace throughout the world, as well as increasing frequency of numerous persistent conditions consisting of cancer cells, autoimmune illness, bone and cartilage diseases are elements anticipated to enhance the market development throughout the forecast period.

The mesenchymal stem cells market is obtaining favorable assistance by the reliable federal government policies, as well as funding for R&D activities which is anticipated to influence the market growth over coming years. According to the reports released by world health organization (WHO), by 2050 individuals aged over 60 will certainly make up greater than 20% of the globe's population. Of that 20%, a traditional quote of 15% is estimated to have symptomatic OA, as well as one-third of these individuals are expected to be influenced by extreme specials needs. Taking into consideration all these aspects, the market for mesenchymal stem cells will certainly witness a substantial development in the future.

Increasing demand for better healthcare facilities, rising geriatric population across the globe, and continuous research and development activities in this area by the key players is expected to have a positive impact on the growth of Mesenchymal Stem Cells market. North America generated the highest revenue in 2017, and is expected to be the leading region globally during the forecast period. The Asia Pacific market is also expected to witness significant market growth in coming years. Developing healthcare infrastructure among countries such as China, India in this region is observed to be the major factor promoting the growth of this market during the forecast period.

The major key players operating in the industry are Cell Applications, Inc., Cyagen Biosciences Inc. Axol Bioscience Ltd., Cytori Therapeutics Inc., Stem cell technologies Inc., Celprogen, Inc. BrainStorm Cell Therapeutics, Stemedica Cell Technologies, Inc. These companies launch new products and undertake strategic collaboration and partnerships with other companies in this market to expand presence and to meet the increasing needs and requirements of consumers.

The marker research report has segmented the global mesenchymal stem cells market on the basis of source type, application and region:

Mesenchymal Stem Cells Source Type Outlook (Revenue, USD Million, 2015 - 2026)

Bone Marrow

Umbilical Cord Blood

Peripheral Blood

Lung Tissue

Synovial Tissues

Amniotic Fluids

Adipose Tissues

Mesenchymal Stem Cells Application Outlook (Revenue, USD Million, 2015 - 2026)

Injuries

Drug Discovery

Cardiovascular Infraction

Others

Request a discount on standard prices of this premium report at: https://www.marketstudyreport.com/check-for-discount/1695294/?utm_source=marketwatch.com&utm_medium=ADS

Table of Contents

1.1. Research goal & scope

1.2. Research assumptions

1.3. Research Methodology

1.3.1. Primary data sources

1.3.2. Secondary data sources

1.4. Key take-away

1.5. Stakeholders

2.1. Market Definition

2.2. Market Segmentation

3.1. Mesenchymal Stem Cells - Industry snapshot

3.2. Mesenchymal Stem Cells - Ecosystem analysis

3.3. Mesenchymal Stem Cells Market Dynamics

3.3.1. Mesenchymal Stem Cells - Market Forces

3.3.2. Mesenchymal Stem Cells Market Driver Analysis

3.3.3. Mesenchymal Stem Cells Market Restraint/Challenges analysis

3.3.4. Mesenchymal Stem Cells Market Opportunity Analysis

3.4. Industry analysis - Porter's five force

3.4.1. Bargaining power of supplier

3.4.2. Bargaining power of buyer

3.4.3. Threat of substitute

3.4.4. Threat of new entrant

3.4.5. Degree of competition

3.5. Mesenchymal Stem Cells Market PEST Analysis

3.6. Mesenchymal Stem Cells Market Value Chain Analysis

3.7. Mesenchymal Stem Cells Industry Trends

3.8. Competitive Ranking Analysis

4.1. Key Findings

4.2. Bone Marrow

4.3. Umbilical Cord Blood

4.4. Peripheral Blood

4.5. Lung Tissue

4.6. Synovial Tissues

4.7. Amniotic Fluids

4.8. Adipose Tissues.

5.1. Key Findings

5.2. Injuries

5.3. Drug Discovery

5.4. Cardiovascular Infraction

5.5. Others

6.1. Key Findings

6.2. North America

6.2.1. U. S.

6.2.2. Canada

6.3. Europe

6.3.1. Germany

6.3.2. UK

6.3.3. France

6.3.4. Italy

6.3.5. Spain

6.3.6. Russia

6.3.7. Rest of Europe

6.4. Asia-Pacific

6.4.1. China

6.4.2. India

6.4.3. Japan

6.4.4. Singapore

6.4.5. Malaysia

6.4.6. Australia

6.4.7. Rest of Asia-Pacific

6.5. Latin America

6.5.1. Mexico

6.5.2. Brazil

6.5.3. Argentina

6.5.4. Rest of LATAM

6.6. Middle East & Africa

7.1. Cell Applications, Inc.

7.1.1. Overview

7.1.2. Financials

7.1.3. Product Benchmarking

7.1.4. Recent Developments

7.2. Cyagen Biosciences Inc.

7.2.1. Overview

7.2.2. Financials

7.2.3. Product Benchmarking

7.2.4. Recent Developments

Originally posted here:

At 7% CAGR, Mesenchymal Stem Cells Market Size, Growth Set ...

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Are Mesenchymal Stem Cells a Promising Treatment for COVID …

A recent pilot study in China in which seven COVID-19 patients received intravenous infusions of donor mesenchymal stem cellsmultipotent cells thought to have immunomodulatory capacitiesindicates that the intervention was safe, and that the approach may improve patient outcomes. While all seven patients recovered, scientists are mixed in their opinions on the logic behind the approach and how well it truly performed.

On Sunday (April 5) the US Food and Drug Administraton approved mesenchymal stem cell (MSC) treatments for use in the very sickest COVID-19 patients under whats known as expanded access compassionate use.

The rationale for [the China] study is not clear [and] the results are . . . inconclusive in terms of how effective it is, says developmental biologist and stem cell researcher Christine Mummery of Leiden University, who has no conflicts of interest to declare. One should view it with a certain amount of healthy skepticism.

Regenerative medicine researcher Ashok Shetty of Texas A&M University College of Medicine disagrees. The results of the study in China demonstrate that intravenous infusion of MSCs is a safe and effective approach for treating patients with COVID-19 pneumonia, including elderly patients displaying severe pneumonia, he writes in an email to The Scientist. However, studies in a larger cohort of patients are needed to validate these benefits. Shetty was not involved with the study and says he does not have any conflicts of interest with companies providing MSCs for therapy, but has previously received project funding from CellTexa company involved in MSC-based therapiesfor unrelated work on Alzheimers disease.

COVID-19, the disease caused by the novel SARS-CoV-2 coronavirus, can have vastly different outcomessome infected individuals are symptom-free, others have a mild, flu-like illness, a smaller number of patients become critically ill with severe pneumonia, and some die. Global deaths currently stand at over 92,000.

For the sickest patients, there appears to be a frequently observed pathologyan uncontrolled ramping up of the immune response, of the sort observed in sepsis, known as cytokine release syndrome or, more colloquially, as a cytokine storm.

Cytokines are small proteins released by immune cells that orchestrate the attack-and-destroy mode of the hosts immune system when faced with a foreign invader. But if levels of these proteins surge wildly, and the immune system goes into overdrive, the patients own tissues and organs can be damagedoften fatally.

The rationale for the Chinese pilot study was that MSCs may help to combat a cytokine storm. MSCs are multipotent cells found in various locations in the body including bone marrow, placenta, and umbilical cord that are reported to have immunodulatory abilities. Indeed, on the basis of this ability, MSCs isolated from donors and expanded in culture are infused into patients as experimental treatments for a number of different diseases. For example, there are trials underway examining the use of MSCs for acute respiratory distress syndrome (ARDS)a build up of fluid on the lungs that results in severe oxygen deprivation. ARDS is a common manifestation of cytokine storms, and the cause of death in many COVID-19 patients.

But the evidence for effective immune response modulation is not that strong, says Mummery. Many of [the trials] have turned out not to be significant in terms of clinical outcome. Theres also a great deal of variability in terms of the source tissue of the MSCs and therefore the type or types of cells that are being injected, she says. And the mechanism of action isnt clear. As to whether they work, she says, you have believers and disbelievers.

An expert in cytokine storms, Randy Cron of the University of Alabama at Birmingham points out that there are other drugs in trials for tackling cytokine storms that are already available, including tocilizumab, which was recently approved in China and the US for the treatment of severe COVID-19 cases. MSCs, which are more experimental, he says, therefore wouldnt be the first thing that comes to my mind [for COVID-19 treatment], but, if it works, it works. Cron has links to certain pharmaceutical companies that manufacture drugs for treating cytokine storms.

In Japan, MSCs have been approved to treat another form of cytokine storm called graft-versus-host disease, and are pending such approval in the US. There are also a number of clinical trials starting to test the benefits of MSCs for treating COVID-19.

Theres a lot of circumstantial evidence that suggests [MSCs] should work . . . in this realm, says Martin Grumet, a stem cell researcher at Rutgers University and the chief scientific officer of CytoStormRx, a company developing technologies for MSC therapies. Grumet, who did not participate in the Chinese study, adds that the data look promising.

In the Chinese study, which was reported in Aging and Disease last month, seven COVID-19 patientsone critically ill, four severely ill and two with milder symptomswere given intravenous infusions of MSCs and, in all cases, the patients recovered with some being discharged from the hospital by the end of the 14-day observation period. In contrast, of the three patients in the placebo control group, all of whom had severe disease, one died, one developed ARDS, and one achieved a stable condition.

The two patients with the worst outcomes (death and ARDS), were about 10 years older than the oldest subjects in the test group, points out Daniel OToole of the National University of Ireland who was not involved in the research. Its very well established that the mortality rate [of COVID-19 patients] is probably more connected to age than anything, he says, indicating this may have skewed the results. OToole has no conflicts of interest to declare.

In addition to these seven patients, a 65-year-old female COVID-19 patient received MSC therapy in a separate case study reported in a paper submitted to the preprint site ChinaXiv at the end of February. Her condition also improved, but, says Cron, the patient, at least by many of the lab markers, was getting better . . . before the mesenchymal stem cell [treatment]. So the result is not compelling, he says.

We understand that it is only a small number of cases, says Kunlin Jin of the University of North Texas Health Science Center who is an author of the Aging and Disease paper. But from the results, he says, we can see that MSCs are a very promising approach for treatment of COVID-19 patients.

Stem cell biologist Paul Knoepfler of the University of California, Davis, writes in an email to The Scientist that he is not convinced at all. The disease is so variable and the study numbers so small that, they dont have the power from a few patients to say anything about efficacy. They dont even really show that the approach is safe, he adds. Because MSCs are thought to suppress immunity, there are also risks . . . that MSCs could weaken the overall immune response to the novel coronavirus, he adds. Knoepfler has no conflicts of interest to declare.

Its a great relief that [following] injection of MSCs into these patients, they didnt suddenly all die, says Mummery. But she agrees with Knoepfler that its too early to determine safety. While MSCs are generally considered safe and well tolerated by patients, we dont know in this particular group of patients what the safety record is.

Doctors are likely to get more data on safety and efficacy soon. Lin tells The Scientist that his team now has unpublished data from a further 24 MSC-treated patientsall of whom, he claims, have improved. And the FDAs recent approval of the treatment (for extreme cases and trials) together with the recruitment of COVID-19 patients to existing MSC trials for ARDS around the world, mean data will likely come in fast. Unfortunately, says OToole, my suspicion is there will be large numbers coming soon, because there probably wont be anything else in the ICUs except for COVID-19 ARDS patients.

Z. Leng et al., Transplantation of ACE2- mesenchymal stem cells improves the outcome of patients with COVID-19 pneumonia,Aging and Disease, 11:21628, 2020.

B. Liang et al., Clinical remission of a critically ill COVID-19 patient treated by human umbilical cord mesenchymal stem cells,ChinaXiv, 202002.00084, 2020.

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Are Mesenchymal Stem Cells a Promising Treatment for COVID ...

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Germline mutation of MDM4, a major p53 regulator, in a familial syndrome of defective telomere maintenance – Science Advances

Abstract

Dyskeratosis congenita is a cancer-prone inherited bone marrow failure syndrome caused by telomere dysfunction. A mouse model recently suggested that p53 regulates telomere metabolism, but the clinical relevance of this finding remained uncertain. Here, a germline missense mutation of MDM4, a negative regulator of p53, was found in a family with features suggestive of dyskeratosis congenita, e.g., bone marrow hypocellularity, short telomeres, tongue squamous cell carcinoma, and acute myeloid leukemia. Using a mouse model, we show that this mutation (p.T454M) leads to increased p53 activity, decreased telomere length, and bone marrow failure. Variations in p53 activity markedly altered the phenotype of Mdm4 mutant mice, suggesting an explanation for the variable expressivity of disease symptoms in the family. Our data indicate that a germline activation of the p53 pathway may cause telomere dysfunction and point to polymorphisms affecting this pathway as potential genetic modifiers of telomere biology and bone marrow function.

TP53 is the gene most frequently mutated in human tumors (1), and germ lineinactivating p53 mutations cause the Li-Fraumeni syndrome of cancer predisposition (2). In addition, accelerated tumorigenesis has been associated with polymorphisms increasing the expression of MDM2 or MDM4, the essential p53 inhibitors (3, 4). Alterations of the p53/MDM2/MDM4 regulatory node are, thus, mainly known to promote cancer. Unexpectedly, however, we recently found that mice expressing p5331, a hyperactive mutant p53 lacking its C terminus, recapitulated the complete phenotype of patients with dyskeratosis congenita (DC) (5).

DC is a telomere biology disorder characterized by the mucocutaneous triad of abnormal skin pigmentation, nail dystrophy, and oral leukoplakia; patients are also at very high risk of bone marrow failure, pulmonary fibrosis, and cancer, especially head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukemia (AML) (6). Patients with DC are known to exhibit disease diversity in terms of age of onset, symptoms, and severity due to the mode of inheritance and causative gene (7, 8). DC is caused by germline mutations in genes encoding key components of telomere biology: the telomerase holoenzyme (DKC1, TERC, TERT, NOP10, and NHP2), the shelterin telomere protection complex (ACD, TINF2, and POT1), telomere capping proteins (CTC1 and STN1), and other proteins interacting with these cellular processes (RTEL1, NAF1, WRAP53, and PARN) (6). Twenty to 30% of affected individuals remain unexplained at the molecular level.

Our finding that p5331/31 mice were remarkable models of DC was initially unexpected for two reasons. First, an increased p53 activity was not expected to cause telomere dysfunction, given the well-accepted notion that p53 acts as the guardian of the genome. However, p53 is now known to down-regulate the expression of many genes involved in genome maintenance (5, 9, 10), and this might actually contribute to its toolkit to prevent tumor formation (11). Second, telomere biology diseases are usually difficult to model in mice because of differences in telomere length and telomerase expression between mice and humans. Mice that lack telomerase exhibited short telomeres only after three or four generations (G3/G4) of intracrosses (12, 13). However, mice with a telomerase haploinsufficiency and a deficient shelterin complex exhibited telomere dysfunction and DC features in a single generation (G1) (14). Because DC features were observed in G1 p5331/31 mice, we supposed that p53 might exert pleiotropic effects on telomere maintenance. Consistent with this, we found that murine p53 down-regulates several genes implicated in telomere biology (5, 9). Because some of these genes were also down-regulated by p53 in human cells (5, 9), our data suggested that an activating p53 mutation might cause features of DC in humans. However, this conclusion remained speculative in the absence of any clinical evidence.

Here, we report the identification of a germline missense mutation in MDM4, encoding an essential and specific negative regulator of p53, in a family presenting some DC-like phenotypic traits. We used a mouse model to demonstrate that this mutation leads to p53 activation, short telomeres, and bone marrow failure. Together, our results provide compelling evidence that a germline mutation affecting a specific p53 regulator may cause DC-like features in both humans and mice.

Family NCI-226 first enrolled in the National Cancer Institute (NCI) inherited bone marrow failure syndrome (IBMFS) cohort in 2008 (Fig. 1A and table S1). At the time, the proband (226-1) was 17 years of age and had a history of neutropenia, bone marrow hypocellularity, vague gastrointestinal symptoms, and chronic pain. His mother (226-4) also had intermittent neutropenia and a hypocellular bone marrow. Notably, his maternal aunt (226-7) had a history of melanoma and died at age 52 because of AML. The maternal aunts daughter (probands cousin, 226-8) had HNSCC at age 27 years, intermittent neutropenia, and bone marrow hypocellularity, while her son (probands cousin, 226-9) was diagnosed with metastatic HNSCC at 42 years of age. The probands father (226-3) was healthy with the exception of hemochromatosis. An IBMFS was suspected on the basis of the family history of cancer and neutropenia. Chromosome breakage for Fanconi anemia was normal, while lymphocyte telomeres were between the 1st and 10th percentiles in the proband and maternal cousin (226-8) (Fig. 1, B and C). The proband was tested for mutations in known DC-causing genes, and a TERT variant (p.W203S) was identified. Unexpectedly, however, the variant was found to be inherited from his father. TERT p.W203S is not present in gnomAD, but it is predicted to be tolerated by MetaSVM (15).

(A) Pedigree of family NCI-226. Arrow indicates proband. Cancer histories include oral squamous cell carcinoma for 226-8 at age 27 years and for 226-9 at age 42 years, and melanoma at 51 years and AML at 52 years for 226-7 (see table S1 for further details). 226-5 had lung cancer at age 69 years. 226-6 had non-Hodgkin lymphoma at age 91 years. In addition, four siblings of 226-6 had cancer: one with breast, two with lung, and one with ovary or uterus (not specified). Sequencing of 226-5, 226-6, 226-7, and 226-9 was not possible because of lack of available DNA. (B and C) Lymphocyte telomere lengths (TL) of study participants. Total lymphocyte telomere lengths are shown and were measured by flow cytometry with in situ hybridization. (B) Graphical depiction of telomere length in relation to age. Four individuals had telomeres measured twice. Legend is in (C). Percentiles (%ile) are based on 400 healthy individuals (50). (C) Age at measurement(s) and telomere length in kilobases. (D) Sequence of the MDM4 RING domain (residues 436 to 490) with secondary structure residues indicated (black boxes). The P-loop motif is highlighted in gray, and the mutated residue in red. (E) The mutant RING domain retains ATP-binding capacity. Wild-type (WT) and mutant (TM) glutathione S-transferase (GST)RING proteins, or GST alone, were incubated with 10 nM ATP and 5 Ci ATP-32P for 10 min at room temperature, filtered through nitrocellulose, and counted by liquid scintillation CPM, counts per minute. Results from two independent experiments. (F) The mutant MDM4 RING domain has an altered capacity to dimerize with the MDM2 RING. Two-hybrid assays were carried out as described (47). -LW, minus leucine and tryptophan; -LWHA, minus leucine, tryptophan, histidine and adenine; OD, optical density. Growth on the -LWHA medium indicates protein interaction, readily observed between MDM2 (M2-BD) and WT MDM4 (M4-AD WT) but faintly visible between MDM2 and MDM4T454M (M4-AD TM). (G) Impact of the mutation in transfected human cells. U2OS cells were transfected with an empty vector (EV) or an expression plasmid encoding a Myc-tagged MDM4 (WT or T454M) protein and then treated or not with cycloheximide (CHX) to inhibit protein synthesis, and protein extracts were immunoblotted with antibodies against Myc, p21, or actin. Bands were normalized to actin, and a value of 1 was assigned to cells transfected with the WT MDM4 expression plasmid (for Myc) or with the empty vector (for p21).

Since the TERT variant did not track with disease inheritance, whole-exome sequencing (WES) was performed to search for a causal gene. The whole-exome data were filtered by maternal autosomal inheritance and revealed three genes with heterozygous missense mutations potentially deleterious according to bioinformatics predictions: MDM4, KRT76, and REM1 (table S2). Given the limited knowledge of the function of KRT76 and REM1, and our prior knowledge of a DC-like phenotype in p5331/31 mice, we chose to focus on the mutation affecting MDM4 because it encodes a major negative regulator of p53. Although the T454M mutation does not affect the p53 interaction domain of MDM4, it might affect p53 regulation because it affects the MDM4 RING domain: Residue 454 is both part of a P-loop motif thought to confer adenosine triphosphate (ATP)binding capacity (16) and part of a strand important for MDM2-MDM4 heterodimerization (Fig. 1D) (17). The mutant RING domain had fully retained its capacity to bind ATP specifically (Fig. 1E and fig. S1A) but exhibited an altered capacity to interact with the MDM2 RING domain in a yeast two-hybrid assay (Fig. 1F). We next used transfection experiments to evaluate the consequences of this mutation on the full-length protein in human cells. We transfected U2OS cellsknown to have a functional but attenuated p53 pathway due to MDM2 overexpression (18)with either an empty vector or an expression plasmid encoding a Myc-tagged MDM4WT or MDM4T454M protein. Compared with cells transfected with the empty vector, cells transfected with a MDM4WT or a MDM4T454M expression plasmid exhibited decreased p21 levels, indicating MDM4-mediated p53 inhibition in both cases (Fig. 1G). However, the decrease in p21 levels was less pronounced in cells expressing MDM4T454M than in cells expressing MDM4WT (Fig. 1G) despite similar transfection efficiencies (fig. S1B). The lower expression levels of the MDM4T454M protein likely contributed to its decreased capacity to inhibit p53 (Fig. 1G). In this experimental setting, the treatment with cycloheximide did not reveal any significant difference in stability between the mutant and wild-type (WT) MDM4 proteins (Fig. 1G and quantification in fig. S1C), raising the possibility that the observed lower MDM4T454M protein levels might result from differences in mRNA translation efficiency. Together, these preliminary results argued for an impact of the mutation on MDM4 function, leading to p53 activation.

The MDM4 RING domain is remarkably conserved throughout evolution, e.g., with 91% identity between the RING domains of human MDM4 and mouse Mdm4 (19). Thus, we decided to create a mouse model to precisely evaluate the physiological impact of the human mutation. We used homologous recombination in embryonic stem (ES) cells to target the p.T454M mutation at the Mdm4 locus (Fig. 2A). Targeted recombinants were identified by long-range polymerase chain reaction (PCR) (Fig. 2B), confirmed by DNA sequencing (Fig. 2C), and the structure of the recombinant allele was further analyzed by Southern blots with probes located 5 and 3 of the targeted mutation (Fig. 2D). Recombinant ES clones were then microinjected into blastocysts to generate chimeric mice, and chimeras were mated with PGK-Cre mice to excise the Neo gene. PCR was used to verify transmission through the germ line of the Mdm4T454M (noted below Mdm4TM) mutation and to genotype the mouse colony and mouse embryonic fibroblasts (MEFs) (Fig. 2E). We first isolated RNAs from Mdm4TM/TM MEFs and sequenced the entire Mdm4 coding sequence: The Mdm4TM sequence was identical to the WT Mdm4 sequence except for the introduced missense mutation (not shown). Furthermore, like its human counterpart, the Mdm4 gene encodes two major transcripts: Mdm4-FL, encoding the full-length oncoprotein that inhibits p53, and Mdm4-S, encoding a shorter, extremely unstable protein (20, 21). We observed, in unstressed cells as well as in cells treated with Nutlin [a molecule that activates p53 by preventing Mdm2-p53 interactions (22) without altering Mdm4-p53 interactions (23, 24)], that the Mdm4TM mutation affected neither Mdm4-FL nor Mdm4-S mRNA levels (Fig. 2F). In Western blots, however, Mdm4-FL was the only detectable isoform, and it was expressed at lower levels in the mutant MEFs (Fig. 2G).

(A) Targeting strategy. Homologous recombination in ES cells was used to target the T454M mutation at the Mdm4 locus. For the Mdm4 WT allele, exons 9 to 11 are shown [black boxes, coding sequences; white box, 3 untranslated region (3UTR)] and Bam HI (BH) restriction sites. Above, the targeting construct contains the following: (i) a 2.9-kb-long 5 homology region encompassing exon 10, intron 10, and exon 11 sequences upstream the mutation; (ii) the mutation (asterisk) within exon 11; (iii) a 2.6-kb-long fragment encompassing the 3 end of the gene and sequences immediately downstream; (iv) a neomycin selection gene (Neo) flanked by loxP sequences (gray arrowheads) and an additional BH site; (v) a 2.1-kb-long 3 homology region containing sequences downstream Mdm4; and (vi) the Diphtheria toxin a gene (DTA) for targeting enrichment. (B to D) screening of G418-resistant ES clones as described in (A), with asterisks (*) indicating positive recombinants: (B) PCR with primers a and b; (C) sequencing after PCR with primers c and d: the sequence for codons 452 to 456 demonstrates heterozygosity at codon 454; (D) Southern blot of Bam HIdigested DNA with the 5 (left) or 3 (right) probe. (E) Examples of fibroblast genotyping by PCR with primers e and f. (F) The Mdm4T454M mutation does not alter Mdm4 mRNA levels. Mdm4-FL (left) and Mdm4-S (right) mRNAs were extracted from WT and Mdm4TM/TM MEFs before or after treatment for 24 hours with 10 M Nutlin, quantified using real-time PCR, and normalized to control mRNAs, and then the value in Nutlin-treated WT MEFs was assigned a value of 1. Results from five independent experiments and >4 MEFs per genotype. ns, not significant in a Students t test. (G) Decreased Mdm4 protein levels in Mdm4TM/TM MEFs. Protein extracts, prepared from MEFs treated as in (F), were immunoblotted with antibodies against Mdm4 or actin. Bands were normalized to actin, and then the values in Nutlin-treated WT cells were assigned a value of 1. p53P/PMdm4E6/E6 MEFs do not express a full-length Mdm4 protein (20): They were loaded to unambiguously identify the Mdm4(-FL) band in the other lanes.

Mdm4TM/TM MEFs contained higher mRNA levels for the p53 targets p21(Cdkn1a) and Mdm2, indicating increased p53 activity (Fig. 3A). Consistent with this, Mdm4TM/TM MEFs exhibited increased p21 and Mdm2 protein levels (Fig. 3B and fig. S2). Moreover, Mdm4TM/TM MEFs prematurely ceased to proliferate when submitted to a 3T3 protocol (Fig. 3C), which also suggests an increased p53 activity. The mean telomere length was decreased by 11% in Mdm4TM/TM MEFs, and a subset of very short telomeres was observed in these cells, hence demonstrating a direct link between the Mdm4TM mutation, p53 activation, and altered telomere biology (Fig. 3D). In p5331/31 MEFs, subtle but significant decreases in expression were previously observed for several genes involved in telomere biology, and in particular, small variations in Rtel1 gene expression were found to have marked effects on the survival of p5331/31 mice (5, 9). Similarly, Mdm4TM/TM MEFs exhibited subtle but significant decreases in expression for Rtel1 and several other genes contributing to telomere biology (Fig. 3E). We previously showed that p53 activation correlates with an increased binding of the E2F4 repressor at the Rtel1 promoter (9). Hence, the decreased Rtel1 mRNA levels in Mdm4TM/TM MEFs most likely resulted from increased p53 signaling. Consistent with this, a further increase in p53 activity, induced by Nutlin, led to further decreases in Rtel1 mRNA and protein levels, in both WT and Mdm4TM/TM cells (fig. S3A). Recently, in apparent contradiction with our finding that p53 activation can cause telomere shortening (5), p53 was proposed to prevent telomere DNA degradation by inducing subtelomeric transcripts, including telomere repeat-containing RNA (TERRA) (25, 26), which suggested a complex, possibly context-dependent impact of p53 on telomeres (27). This led us to compare TERRA transcripts in WT and Mdm4TM/TM cells. Consistent with an earlier report (26), p53 activation led to increased TERRA at the mouse Xq subtelomeric region in WT cells (fig. S3B). However, Mdm4TM/TM cells failed to induce TERRA in response to stress (fig. S3B). Together, our data suggest that the telomere shortening observed in Mdm4TM/TM cells results from a p53-dependent decrease in expression of several telomere-related genes and, notably, Rtel1, a gene mutated in several families with DC (6). In addition, although evidence that altered TERRA levels can cause DC is currently lacking, we cannot exclude that an altered regulation of TERRA expression might contribute to telomere defects in Mdm4TM/TM cells.

(A) Quantification of p21 and Mdm2 mRNAs extracted from WT, Mdm4+/TM, and Mdm4TM/TM MEFs, treated or not for 24 hours with 10 M Nutlin. mRNA levels were quantified using real-time PCR and normalized to control mRNAs, and then the value in Nutlin-treated WT MEFs was assigned a value of 1. Results from 10 independent experiments. (B) Protein extracts, prepared from p53/, WT, and Mdm4TM/TM MEFs treated as in (A), were immunoblotted with antibodies against Mdm2, Mdm4, p53, p21, or actin. Bands were normalized to actin, and then the values in Nutlin-treated WT MEFs were assigned a value of 1. (C) Proliferation of MEFs in a 3T3 protocol. Each point is the average value of three independent MEFs. (D) Decreased telomere length in Mdm4TM/TM MEFs, as measured by quantitative FISH with a telomeric probe. Results from two MEFs per genotype, and 68 to 75 metaphases per MEF [means + 95% confidence interval (CI) are shown in yellow]. a.u., arbitrary units. (E) Telomere-related genes down-regulated in Mdm4TM/TM MEFs. mRNAs were extracted from unstressed WT and Mdm4TM//TM MEFs, quantified using real-time PCR, and normalized to control mRNAs, and the value in WT MEFs was assigned a value of 1. Results from >3 independent experiments and two MEFs per genotype. In relevant panels: P = 0.08, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Students t (A, C at passage 7, and E) or Mann-Whitney (D) statistical tests.

Mdm4TM/TM mice were born in Mendelian proportions from Mdm4+/TM intercrosses (Fig. 4A) but were smaller than their littermates and died within 0 to 30 min after birth, with signs of severe respiratory distress (Fig. 4, B and C). Consistent with this, Mdm4TM/TM pups at postnatal day 0 (P0) appeared hypoxic (Fig. 4C), and their lungs were very small and dysfunctional (Fig. 4D). Thus, Mdm4TM/TM pups most likely died from neonatal respiratory failure. Tissues from Mdm4TM/TM pups exhibited increased p21 mRNA levels, suggesting an increase in p53 activity in these animals (fig. S4). We next used flowFISH (fluorescence in situ hybridization) with a telomere-specific probe to evaluate the impact of the mutation on telomere length in vivo. Lung cells from Mdm4TM/TM pups (and control G3 Terc/ mice) exhibited a 25% decrease in mean telomere length compared with cells from WT or Mdm4+/TM littermates, indicating altered telomere biology in G1 homozygous mutants (Fig. 4E). Notably, p53 loss or haploinsufficiency rescued the perinatal lethality of Mdm4TM/TM pups, illustrating that the premature death of Mdm4TM/TM mice likely resulted from increased p53 activity (Fig. 4F). However, p53/ and Mdm4TM/TM p53/ mice exhibited similar survival curves, with a fraction of the mice (respectively 4 of 12 and 1 of 6) succumbing to thymic lymphoma in less than 180 days. In contrast, after 180 days, all the p53+/ mice remained alive, whereas most Mdm4TM/TM p53+/ mice had died. Mdm4TM/TM p53+/ mice were smaller than their littermates (Fig. 4G) and exhibited hyperpigmentation of the footpads (Fig. 4H), and 120-day-old Mdm4TM/TM p53+/ mice exhibited abnormal hemograms (Fig. 4I). Furthermore, the Mdm4TM/TM p53+/ mice that died 60 to 160 days after birth exhibited bone marrow hypocellularity (Fig. 4J), indicating bone marrow failure as the likely cause for their premature death.

(A) Mendelian distribution of the offspring from 8 Mdm4+/TM intercrosses. (B) Mdm4TM/TM mice die at birth. Cohort sizes are in parentheses. (C) Mdm4TM/TM neonates are smaller than their littermates and appear hypoxic. (D) Lungs from Mdm4TM/TM P0 pups are hypoplastic and sink in phosphate-buffered saline owing to a lack of air inflation. (E) Flow-FISH analysis of P0 lung cells with a telomere-specific peptide nucleic acid (PNA) probe. Top: Representative results from a WT, a Mdm4+/TM, a Mdm4TM/TM, and a G3 Terc/ mouse are shown. Right: Green fluorescence (fluo.) with black histograms for cells without the probe (measuring cellular autofluorescence) and green histograms for cells with the probe. The shift in fluorescence intensity is smaller in Mdm4TM/TM and Terc/ cells (c or d < a or b), indicating reduced telomere length. Left: Propidium iodide (PI) fluorescence histograms are superposed for cells with or without the probe. Below: Statistical analysis of green fluorescence shifts (see Materials and Methods). Means + 95% CI are shown; data are from two to three mice and >3800 cells per genotype. (F) Impact of decreased p53 activity on Mdm4TM/TM animals. Cohort sizes are in parentheses. (G) Examples of littermates with indicated genotypes. (H) Hind legs of mice with indicated genotypes. (I) Mdm4TM/TM p53+/ mice exhibit abnormal hemograms. Counts for white blood cells (WBC), red blood cells (RBC), and platelets (PLT) for age-matched (120 days old) animals are shown. (J) Hematoxylin and eosin staining of sternum sections from WT and Mdm4TM/TM p53+/ mice. In relevant panels: ns, not significant; *P < 0.05, ***P < 0.001, and ****P < 0.0001 by Mantel-Cox (B and F), Students t (C, D, G, and I), or Mann-Whitney (E) statistical tests. Photo credits: E.T. and R.D., Institut Curie (C, G, and H); R.D., Institut Curie (D).

Although Mdm4TM/TM MEFs and mice were useful to demonstrate that the Mdm4T454M mutation leads to p53 activation and short telomeres, a detailed analysis of Mdm4+/TM mice appeared more relevant to model the NCI-226 family, in which all affected relatives were heterozygous carriers of the MDM4T454M mutation. Unlike Mdm4TM/TM mice, most Mdm4+/TM animals remained alive 6 months after birth and had no apparent phenotype, similarly to WT mice (Fig. 5A). This was consistent with our analyses in fibroblasts because Mdm4+/TM MEFs behaved like WT cells in a 3T3 proliferation assay (Fig. 3C). However, p53 target genes appeared to be transactivated slightly more efficiently in Mdm4+/TM than in WT cells (Fig. 3A), and 30% of Mdm4+/TM mice exhibited a slight hyperpigmentation of the footpads, suggesting a subtle increase in p53 activity (Fig. 5B). We reasoned that a further, subtle increase in p53 activity might affect the survival of Mdm4+/TM mice. We tested this hypothesis by mating Mdm4+/TM animals with p53+/31 mice. p53+/31 mice were previously found to exhibit a slight increase in p53 activity and to remain alive for over a year (5). Notably, unlike Mdm4+/TM or p53+/31 heterozygous mice, Mdm4+/TM p53+/31 compound heterozygotes died in less than 3 months (Fig. 5A) and exhibited many features associated with strong p53 activation. Mdm4+/TM p53+/31 mice exhibited intense skin hyperpigmentation (Fig. 5C), were much smaller than their littermates (Fig. 5D), and exhibited heart hypertrophy (Fig. 5E) and thymic hypoplasia (Fig. 5F) and the males had testicular hypoplasia (Fig. 5G). Bone marrow failure was the likely cause for the premature death of Mdm4+/TM p53+/31 mice, as indicated by abnormal hemograms of 18-day-old (P18) compound heterozygotes (Fig. 5H) and bone marrow hypocellularity in the sternum sections of moribund Mdm4+/TM p53+/31 animals (Fig. 5I). We next used flow-FISH to analyze telomere length in the bone marrow cells of P18 WT, Mdm4+/TM, p53+/31, and Mdm4+/TM p53+/31 mice. We found no significant difference between telomere lengths in cells from five WT and three Mdm4+/TM mice with normal skin pigmentation, whereas cells from two Mdm4+/TM mice with increased skin pigmentation (or from p53+/31 mice) exhibited marginal (5 to 7%) decreases in mean telomere length. Notably, in G1 Mdm4+/TM p53+/31 cells, the average telomere length was decreased by 34% (Fig. 5J). Together, these results demonstrate that Mdm4+/TM mice are hypersensitive to subtle increases in p53 activity. Consistent with this, Mdm4+/TM p53+/31 MEFs also exhibited increased p53 signaling and accelerated proliferation arrest in a 3T3 protocol (fig. S5). In sum, the comparison between Mdm4TM/TM and Mdm4TM/TM p53+/ mice, or between Mdm4+/TM and Mdm4+/TM p53+/31 animals, indicated that subtle variations in p53 signaling had marked effects on the phenotypic consequences of the Mdm4T454M mutation (table S3).

(A) Impact of increased p53 activity on Mdm4+/TM animals. Cohort sizes are in parentheses. (B) Footpads from Mdm4+/TM mice appear normal (top) or exhibit a subtle increase in pigmentation (bottom). (C) Mdm4+/TM p53+/31 mice exhibit strong skin hyperpigmentation. (D) Mdm4+/TM p53+/31 mice are smaller than age-matched WT mice. (E to G) Mdm4+/TM p53+/31 mice exhibit heart hypertrophy (E) as well as thymic (F) and testicular (G) hypoplasia. (H) Mdm4+/TM p53+/31 mice exhibit abnormal hemograms. Counts for white blood cells, red blood cells, and platelets for five age-matched (P18) animals per genotype are shown. (I) Hematoxylin and eosin staining of sternum sections from mice of the indicated genotypes. (J) Flow-FISH analysis of P18 bone marrow cells with a telomere-specific PNA probe. Top: Representative results for a WT, a Mdm4+/TM with normal skin pigmentation (nsp), a Mdm4+/TM with increased footpad skin pigmentation (isp), a p53+/31, and a Mdm4+/TM p53+/31 mouse are shown; black histograms, cells without the probe; green histograms, cells with the probe. The smallest shift in fluorescence intensity (e) was observed with Mdm4+/TM p53+/31 cells. Bottom: Statistical analysis of green fluorescence shifts. Means + 95% CI are shown; data are from >1500 cells per genotype. In relevant panels: ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Mantel-Cox (A), Students t (D and E to H), or Mann-Whitney (J) statistical tests. Photo credits: R.D. and P.L., Institut Curie (B); E.T. and R.D., Institut Curie (C and D).

The carriers of the MDM4T454M mutation exhibited considerable heterogeneity in their phenotypes (Fig. 1 and table S1). The data from our mouse model suggested that variations in p53 activity might account for the variable expressivity and penetrance of clinical features among the NCI-226 MDM4+/T454M relatives. Hence, we analyzed nine known common polymorphisms reported to affect p53 activity and tumorigenesis (four at the TP53 locus, two at the MDM2 locus, and three at the MDM4 locus) (3,4,2832). Among the four MDM4+/T454M relatives, the proband (NCI-226-1) is more difficult to interpret because the potential contribution of the TERT p.W203S variant to his phenotype cannot be ruled out (even though it appears unlikely according to in silico predictions). The MDM4 allele encoding the mutant protein (p.T454M) appears associated with the C allele of single-nucleotide polymorphism (SNP) rs4245739, the G allele of SNP rs11801299, and the G allele of SNP rs1380576 (Fig. 6A). These three MDM4 variant alleles are associated with increased p53 activity (4,32) and might, thus, synergize with the MDM4T454M mutation in this family.

(A) Genotyping of polymorphisms that may affect the p53 pathway. The SNPs rs1800371 and rs1042522 modify the p53 protein sequence (28,29), whereas rs17878362 and rs17880560 are singlets (A1) or doublets (A2) of G-rich sequences in noncoding regions of TP53 that affect p53 expression (30). SNPs rs117039649 and rs2279744, in the MDM2 promoter, affect MDM2 mRNA levels (3,31). Three SNPs are at the MDM4 locus: rs4245739 in the 3UTR region affects MDM4 protein levels (4), whereas rs11801299 and rs1380576 were associated with an increased risk of developing retinoblastoma (32), a cancer type with frequent MDM4 alterations (51). Polymorphisms that differ among family members are in bold, with the allele (or haplotype) associated with increased p53 activity in green (because it may synergize with the effects of the MDM4T454M mutation). Alleles (or haplotypes) for which there is evidence of decreased p53 activity, or for which the effect is uncertain, are highlighted in red or blue, respectively. Please note that the clinical effects of the TP53 rs1042522 SNP have recently been contested (33), so that all alleles for this SNP were labeled in blue. MAF, minor allele frequency reported for all gnomAD populations combined. https://gnomad.broadinstitute.org (52). (B) Comparative analysis of primary fibroblasts from family members 226-4 and 226-8. p21 and RTEL1 mRNAs, extracted from cells from relatives NCI 226-4 and NCI 226-8 or two unrelated patients with DC carrying a TINF2 or a TERT mutation, were quantified using real-time PCR, normalized to control mRNAs, and then expressed relative to the mean values in TINF2 and TERT mutant cells. ns, not significant, **P < 0.01 and ***P < 0.001 in a Students t test.

The probands affected cousin (226-8) exhibited a very early onset of disease, with lymphocyte telomere length within or below the first percentile of age-matched control participants and tongue squamous cell carcinoma at age 27 (Fig. 1 and table S1). The WT MDM4 allele of 226-8 carried the rs4245739 C, the rs11801299 G, and the rs1380576 G variants associated with increased p53 activity. This suggests a potential disease-modifying effect of these MDM4 SNPs. In contrast, the probands mother (226-4) was much less severely affected, with telomere length between the 10th and 50th percentiles (Fig. 1). Although we cannot rule out that disease anticipation might contribute to her milder phenotype, note that her WT MDM4 allele carried variants that might correlate with decreased p53 activity and could antagonize the MDM4T454M mutation (rs4245739 A, rs11801299 A, and rs1380576 C; Fig. 6A). Family members 226-4 and 226-8 shared the same genotypes for all the other tested variants, except for TP53 rs1042522, a SNP first reported to affect apoptotic or cell cycle arrest responses (28), but with a clinical effect that now appears controversial (33). The probands sister (226-2), with a B cell deficiency and telomere lengths around the 10th percentile, also appeared less affected than 226-8. All the tested variants at the MDM2 and MDM4 loci were identical between 226-2 and 226-8. However, unlike 226-8, 226-2 exhibited a TP53 allele with an A1A1 haplotype for variants rs17878362 and rs17880560 that might decrease p53 activity (30) and antagonize the effects of the MDM4T454M mutation (Fig. 6A).

We had primary fibroblasts available for two of these family members, 226-4 and 226-8, allowing us to directly assess the functional effect of the MDM4T454M variant in these cells. These fibroblasts were grown in parallel with primary fibroblasts from patients with DC carrying either a TINF2K280E mutation or a TERTP704S mutation, and mRNA levels for p21 and RTEL1 were quantified. In agreement with the notion that a MDM4T454M heterozygous mutation activates p53 signaling in NCI-226 family members, fibroblasts from both 226-4 and 226-8 exhibited increased p21 mRNA levels compared with TINF2 or TERT mutant cells (Fig. 6B). However, cells from 226-4 only exhibited a 2-fold increase in p21 levels, whereas a 12-fold increase was observed for cells from 226-8, consistent with the notion that SNPs affecting the p53 pathway might counteract (for 226-4) or strengthen (for 226-8) the effect of the MDM4T454M mutation. Furthermore, we previously showed that RTEL1 mRNA levels are down-regulated upon p53 activation in human cells (5). RTEL1 mRNA levels appeared normal in cells from 226-4 but were markedly decreased in cells from 226-8, raising the possibility that a threshold in p53 activation might be required to affect RTEL1 expression (Fig. 6B).

Although MDM4 is primarily known for its clinical relevance in cancer biology, our study shows that a germline missense MDM4 mutation may cause features suggestive of DC. In humans, the MDM4 (p.T454M) mutation was identified in this family with neutropenia, bone marrow hypocellularity, early-onset tongue SCC, AML, and telomeres between the 1st and 10th percentiles in the younger generation. In mice, the same Mdm4 mutation notably correlated with increased p53 activity, short telomeres, and bone marrow failure. In both human transfected cells and MEFs, the mutant protein was expressed at lower levels than its WT counterpart, likely contributing to increased p53 activity. Together, these results demonstrate the importance of the MDM4/p53 regulatory axis on telomere biology and DC-like features in both species. Notably, p5331/31 mice were previously found to phenocopy DC (5), but whether this finding was relevant to human disease had remained controversial. When a mutation in PARN was found to cause DC (34), it first appeared consistent with the p5331 mouse model because PARN, the polyadenylate-specific ribonuclease, had been proposed to regulate p53 mRNA stability (35). However, whether PARN regulates the stability of mRNAs is now contested (36). Rather, PARN would regulate the levels of over 200 microRNAs, of which only a few might repress p53 mRNA translation (37). Furthermore, PARN regulates TERC, the telomerase RNA component (38), and TERC overexpression increased telomere length in PARN-deficient cells (39). Thus, whether a germline mutation that specifically activates p53 can cause DC-like features remained to be demonstrated in humans, and our report provides compelling evidence for this, because unlike PARN, MDM4 is a very specific regulator of p53.

A germline antiterminating MDM2 mutation was recently identified in a patient with a Werner-like syndrome of premature aging. Although multiple mechanisms might contribute to the clinical features in that report, a premature cellular senescence resulting from p53 hyperactivation was proposed to play a major role in his segmental progeroid phenotype (40). In that regard, our finding that increased p53 activity correlates with short telomeres appears relevant because telomere attrition is a primary hallmark of aging, well known to trigger cellular senescence (41). Furthermore, germline TP53 frameshift mutations were recently reported in two patients diagnosed with pure red blood cell aplasia and hypogammaglobulinemia, resembling but not entirely consistent with Diamond Blackfan anemia (DBA) (42). In addition to the pure red cell aplasia diagnostic of DBA, those patients were found to exhibit relatively short telomeres (although not as short as telomeres from patients with DC), which may also seem consistent with our results. Our finding of an MDM4 missense mutation in a DC-like family, together with recent reports linking an antiterminating MDM2 mutation to a Werner-like phenotype and TP53 frameshift mutations to DBA-like features, indicates that the clinical impact of germline mutations affecting the p53/MDM2/MDM4 regulatory network is just emerging. An inherited hyperactivation of the p53 pathwayvia a germline TP53, MDM2, or MDM4 mutationmay thus cause either DBA, Werner-like, or DC-like features, but additional work will be required to determine whether mutations in any of these three genes can cause any of these three syndromes. Likewise, several mouse models have implicated p53 deregulation in features of other developmental syndromes including the CHARGE, Treacher-Collins, Waardenburg, or DiGeorge syndrome (43), and it will be important to know whether germline mutations in TP53, MDM2, or MDM4 may cause these additional syndromes in humans.

Heterozygous Mdm4+/TM mice appeared normal but were hypersensitive to variations in p53 activity, and, perhaps most notably, Mdm4+/TM p53+/31 compound heterozygous mice rapidly died from bone marrow failure. Thus, the p5331 mutation acted as a strong genetic modifier of the Mdm4TM mutation. It is tempting to speculate that similarly, among the NCI-226 family members heterozygous for the MDM4T454M allele, differences in the severity of phenotypic traits (e.g., lymphocyte telomere length and bone marrow cellularity) may result, in part, from modifiers affecting the p53 pathway and synergize or antagonize with the effects of the MDM4T454M mutation. To search for potentially relevant modifiers, we looked at nine polymorphisms at the TP53, MDM2, and MDM4 loci that were previously reported to affect p53 activity. Notably, we found that the family member most severely affected (226-8, the probands cousin) carried a TP53 haplotype, as well as SNPs on the WT MDM4 allele, that might synergize with the effects of the MDM4T454M mutation. Conversely, a TP53 haplotype for the probands sister (226-2), or SNPs at the WT MDM4 locus for the probands mother (226-4), might antagonize the impact of MDM4T454M allele. Consistent with this, primary fibroblasts from 226-4 and 226-8 exhibited increased p53 activity, but p53 activation was much stronger in cells from 226-8. Our data, thus, appear consistent with the existence of genetic modifiers at the TP53 and MDM4 loci that may affect DC-like phenotypic traits among family members carrying the MDM4 (p.T454M) mutation. However, this remains speculative given the small number of individuals that could be analyzed. Furthermore, nonexonic variants affecting other genes might also contribute to DC-like traits (44). Last, the TP53 and MDM4 polymorphisms considered here were previously evaluated for their potential impact on tumorigenic processes, rather than DC-like traits such as telomere length or bone marrow hypocellularity. Our data suggest that polymorphisms at the TP53 and MDM4 (and possibly MDM2) loci should be evaluated for their potential impact on bone marrow function and telomere biology.

The individuals in this study are participants in an Institutional Review Boardapproved longitudinal cohort study at the NCI entitled Etiologic Investigation of Cancer Susceptibility in Inherited Bone Marrow Failure Syndromes (www.marrowfailure.cancer.gov, ClinicalTrials.gov NCT00027274) (7). Patients and their family members enrolled in 2008 and completed detailed family history and medical history questionnaires. Detailed medical record review and thorough clinical evaluations of the proband, his sister, parents, and maternal cousin were conducted at the National Institutes of Health (NIH) Clinical Center. Telomere length was measured by flow cytometry with in situ hybridization (flow-FISH) (45) in leukocytes of all patients and family members reported. DNA was extracted from whole blood using standard methods. DNA was not available from 226-7 or 226-9 (Fig. 1). Given the time frame of participant enrollment, Sanger sequencing of DKC1, TINF2, TERT, TERC, and WRAP53 was performed first, followed by exome sequencing.

WES of blood-derived DNA for family NCI-226 was performed at the NCIs Cancer Genomics Research Laboratory as previously described (46). Exome enrichment was performed with NimbleGens SeqCap EZ Human Exome Library v3.0 + UTR (Roche NimbleGen Inc., Madison, WI, USA), targeting 96 Mb of exonic sequence and the flanking untranslated regions (UTRs) on an Illumina HiSeq. Annotation of each exome variant locus was performed using a custom software pipeline. WES variants of interest were identified if they met the following criteria: heterozygous in the proband, his mother, and maternal cousin; nonsynonymous; had a minor allele frequency <0.1% in the Exome Aggregation Consortium databases; and occurred <5 times in our in house database of 4091 individuals. Variants of interest were validated to rule out false-positive findings using an Ion 316 chip on the Ion PGM Sequencer (Life Technologies, Carlsbad, CA, USA).

Primers flanking the MDM4 RING domain were used to amplify RING sequences, and PCR products were cloned (or cloned and mutagenized) in the pGST-parallel2 plasmid. Glutathione S-transferase (GST) fusion proteins were expressed in BL21 (DE3) cells. After induction for 16 hours at 20C with 0.2 mM IPTG (isopropyl--d-thiogalactopyranoside), soluble proteins were extracted by sonication in lysis buffer [50 mM tris (pH 7.0), 300 mM LiSO4, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.2% NP-40, complete Protease inhibitors (Roche) 1]. The soluble protein fraction was incubated with Glutathione Sepharose beads (Pharmacia) at 4C for 2 hours, and the bound proteins were washed with 50 mM tris (pH 7.0), 300 mM LiSO4, and 1 mM DTT and then eluted with an elution buffer [50 mM tris-HCl (pH 7.5), 300 mM NaCl, 1 mM DTT, and 15 mM glutathione]. WT and mutant GST-RING proteins (0, 1, 2, 4, or 8 g) or GST alone (0 or 8 g) was incubated with 10 nM ATP and 5 Ci ATP-32P for 10 min at room temperature, filtered through nitrocellulose, and counted by liquid scintillation. Alternatively, 7 g of either WT or mutant GST-RING proteins was incubated with 5 Ci ATP-32P for 10 min at room temperature and increasing amounts (0, 0.02, 2, 20, and 200 M) of ATP or guanosine triphosphate (GTP), filtered through nitrocellulose, and counted by liquid scintillation.

The yeast two-hybrid assays were performed as described (47). Briefly, MDM4 and MDM2 RING open reading frames were cloned in plasmids derived from the two-hybrid vectors pGADT7 (Gal4-activating domain) and pGBKT7 (Gal4-binding domain) creating N-terminal fusions and transformed in yeast haploid strains Y187 and AH109 (Clontech). Interactions were scored, after mating and diploid selection on dropout medium without leucine and tryptophan, as growth on dropout medium without leucine, tryptophan, histidine, and adenine.

U2OS cells (106) were transfected by using Lipofectamine 2000 (Invitrogen) with pCDNA3.1 (6 g), or 5 106 cells were transfected with 30 g of pCDNA3.1-MycTag-MDM4WT or pCDNA3.1-MycTag-MDM4TM. Twenty-four hours after transfection, cells were treated with cycloheximide (50 g/ml; Sigma-Aldrich, C4859), then scratched in phosphate-buffered saline (PBS) after 2, 4, or 8 hours, pelleted, and snap frozen in liquid nitrogen before protein or RNA extraction with standard protocols.

The targeting vector was generated by recombineering from the RP23-365M5 BAC (bacterial artificial chromosome) clone (CHORI BACPAC Resources) containing mouse Mdm4 and downstream sequences of C57Bl6/J origin. A loxP-flanked neomycin cassette (Neo) and a diphtheria toxin gene (DTA) were inserted downstream of the Mdm4 gene, respectively, for positive and negative selections, and a single-nucleotide mutation encoding the missense mutation T454M (TM) was targeted in the exon 11 of Mdm4. The targeting construct was fully sequenced before use.

CK-35 ES cells were electroporated with the targeting construct linearized with Not I. Recombinant clones were identified by long-range PCR, confirmed by Southern blot, PCR, and DNA sequencing (primer sequences in table S4). Two independent recombinant clones were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping their offspring. Reverse transcription PCR (RT-PCR) of RNAs from Mdm4TM/TM MEFs showed that the mutant complementary DNA (cDNA) differed from an Mdm4 WT sequence only by the engineered missense mutation. The genotyping of p53+/, p53+/31, and G3 Terc/ mice was performed as previously described (5, 12). All experiments were performed according to Institutional Animal Care and Use Committee regulations.

MEFs isolated from 13.5-day embryos were cultured in a 5% CO2 and 3% O2 incubator, in Dulbeccos modified Eagles medium GlutaMAX (Gibco), with 15% fetal bovine serum (Biowest), 100 M 2-mercaptoethanol (Millipore), 0.01 mM Non-Essential Amino Acids, and penicillin/streptavidin (Gibco) for five or fewer passages, except for 3T3 experiments, performed in a 5% CO2 incubator for seven passages. Cells were treated for 24 hours with 10 M Nutlin 3a (Sigma-Aldrich) (22) or 15 M cisplatin (Sigma-Aldrich). Primary human fibroblasts at low passage (p.2 for TINF2K280E, p.3 for NCI-226-4 and NCI-226-8, and p.4 for TERTP704S) were thawed and cultured in fibroblast basal medium (Lonza) with 20% fetal calf serum, l-glutamin, 10 mM Hepes, penicillin/streptavidin, and gentamicin before quantitative PCR (qPCR) analysis.

Total RNA, extracted using NucleoSpin RNA II (Macherey-Nagel), was reverse transcribed using SuperScript IV (Invitrogen), with, for TERRA quantification, a (CCCTAA)4 oligo as described (48). Real-time qPCRs were performed with primer sequences as described (5, 9, 48) on a QuantStudio using Power SYBR Green (Applied Biosystems).

Protein detection by immunoblotting was performed using antibodies against Mdm2 (4B2), Mdm4 (M0445; Sigma-Aldrich), p53 (AF1355, R&D Systems), actin (A2066; Sigma-Aldrich), p21 (F5; Santa Cruz Biotechnology), Myc-Tag (SAB2702192; Sigma-Aldrich), and Rtel1 (from J.-A.L.-V.). Chemiluminescence revelation was achieved with SuperSignal West Dura (Perbio). Bands of interest were quantified by using ImageJ and normalized with actin.

Cells were treated with colcemide (0.5 g/ml) for 1.5 hours, submitted to hypotonic shock, fixed in an (3:1) ethanol/acetic acid solution, and dropped onto glass slides. Quantitative FISH was then carried out as described (5) with a TelC-Cy3 peptide nucleic acid (PNA) probe (Panagene). Images were acquired using a Zeiss Axioplan 2, and telomeric signals were quantified with iVision (Chromaphor).

Flow-FISH with mouse cells was performed as described (45). For each animal, either the lungs were collected or the bone marrow from two tibias and two femurs was collected and red blood cells were lysed; then, 2 106 cells were fixed in 500 l of PNA hybridization buffer [70% deionized formamide, 20 mM tris (pH 7.4), and 0.1% Blocking reagent; Roche] and stored at 20C. Either nothing (control) or 5 l of probe stock solution was added to cells [probe stock solution: 10 M TelC-FAM PNA probe (Panagene), 70% formamide, and 20 mM tris (pH 7.4)], and samples were denatured for 10 min at 80C before hybridization for 2 hours at room temperature. After three washes, cells were resuspended in PBS 1, 0.1% bovine serum albumin, ribonuclease A (1000 U/ml), and propidium iodide (12.5 g/ml) and analyzed with an LSR II fluorescence-activated cell sorter. WT and G3 Terc/ mice were included in all flow-FISH experiments, respectively, as controls of normal and short telomeres. For fluorescence shift analyses, the green histograms (corresponding to cells with the telomeric probe) were sliced into 18 windows of equal width and numbered 0 to 17 according to their distance from the median value in cells without the probe, and the number of cells in each window was quantified with ImageJ. The data from two to five mice per genotype were typically used to calculate mean telomere lengths, expressed relative to the mean in WT cells.

Organs were fixed in formol 4% for 24 hours and then ethanol 70% and embedded in paraffin wax. Serial sections were stained with hematoxylin and eosin using standard procedures (49). For hemograms, 100 l of blood from each animal was recovered retro-orbitally in a 10-l citrate-concentrated solution (S5770; Sigma-Aldrich) and analyzed using an MS9 machine (Melet Schloesing Laboratoires).

DNA extracted from Epstein-Barr virustransformed lymphocytes of NCI-226 family members was amplified with primers flanking nucleotide polymorphisms of interest (primer sequences in table S5), and then PCR products were analyzed by Sanger DNA sequencing.

Analyses with Students t, Mann-Whitney, or Mantel-Cox statistical tests were performed by using GraphPad Prism, and values of P < 0.05 were considered significant.

This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Acknowledgments: We are grateful to the family for valuable contributions to this study. We thank I. Grandjean, C. Caspersen, A. Fosse, and M. Garcia from the Animal Facility, C. Alberti and C. Roulle from the Transgenesis Platform, M. Richardson and A. Nicolas from the Pathology Service, and Z. Maciorowski from the Cell-Sorting Facility of the Institut Curie. We thank A. Chor for help with qPCRs, A. Pyanitskaya, C. Adam, V. Borde, M. Schertzer, and M. Perderiset for plasmids and technical advices, and A. Fajac for comments on the manuscript. F.T. would like to acknowledge the talent, kindness, and loyal support of I. Simeonova and S.J., two exceptional PhD students whose pioneering work led to this study. Funding: The Genetics of Tumor Suppression laboratory received funding from the Ligue Nationale contre le Cancer (Labellisation 2014-2018 and Comit Ile-de-France), the Fondation ARC and the Gefluc. PhD students were supported by fellowships from the Ministre de lEnseignement Suprieur et de la Recherche (to S.J., E.T., and R.D.), the Ligue Nationale contre le Cancer (to S.J.), and the Fondation pour la Recherche Mdicale (to E.T.). The work of S.A.S., N.G., and B.P.A. was supported by the intramural research program of the Division of Cancer Epidemiology and Genetics, NCI, and the NIH Clinical Center. Author contributions: V.L. created the Mdm4T454M mouse model, genotyped mouse cohorts, and performed transfections, yeast two-hybrid assays, protein purifications, and molecular cloning. E.T., R.D., and V.L. managed mouse colonies. E.T., R.D., and P.L. performed mouse anatomopathology. I.D., E.T., R.D., F.T., and J.-A.L.-V. determined mouse telomere lengths. V.L. and S.J. genotyped human polymorphisms and analyzed human fibroblasts. E.T. and R.D. genotyped MEFs and performed 3T3 assays. V.L., R.D., and E.T. performed Western blots. E.T., R.D., V.L., S.J., and P.L. performed qPCRs. B.B. and V.L. performed ATP-binding assays. B.P.A. supervised the NCI IBMFS study. N.G. and S.A.S. evaluated study participants. S.A.S. analyzed the exome sequencing data. F.T. and S.A.S. supervised the project and wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. The human samples can be provided by S.A.S. pending scientific review and a completed material transfer agreement. Requests for human cells should be submitted to S.A.S.

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Germline mutation of MDM4, a major p53 regulator, in a familial syndrome of defective telomere maintenance - Science Advances

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Stem Cell Banking Storage Market Size Analysis, Top Manufacturers, Shares, Growth Opportunities and Forecast to 2026 – Science In Me

New Jersey, United States:The new report has been added by Market Research Intellect to provide a detailed overview of the Stem Cell Banking Storage Market. The study will help to better understand the Stem Cell Banking Storage industry competitors, the sales channel, Stem Cell Banking Storage growth potential, potentially disruptive trends, Stem Cell Banking Storage industry product innovations and the value / volume of size market (regional / national level, Stem Cell Banking Storage- Industrial segments), market share of the best actors / products.

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The report examines the competitive environment scenario observed with key players in Stem Cell Banking Storage sales, the profile of their business, their earnings, their sales, their business tactics, and the forecasting situations of the Stem Cell Banking Storage sales industry. According to studies, the Stem Cell Banking Storage sales market is very competitive and diverse due to global and local suppliers.

The Stem Cell Banking Storage Sales Market Report mainly contains the following Manufacturers:

Market Competition

The competitive landscape of the Stem Cell Banking Storage market is examined in detail in the report, with a focus on the latest developments, the future plans of the main players and the most important growth strategies that they have adopted. The analysts who compiled the report have created a portrait of almost all of the major players in the Stem Cell Banking Storage market, highlighting their key commercial aspects such as production, areas of activity and product portfolio. All companies analyzed in the report are examined on the basis of important factors such as market share, market growth, company size, production, sales and earnings.

Report Highlights

Assessment of sales channels

innovation trends

sustainability strategies

Niche market trends

Market entry analysis

market size and forecast

The geographic department provides data that give you an overview of the turnover of companies and sales figures for the growth activity Stem Cell Banking Storage for electrical meters. Here are the strengths of the geographic divisions: North America (United States, Canada and Mexico), Europe (Germany, Spain, France, Great Britain, Russia and Italy and more), Asia-Pacific (China, Japan, Korea, India and Southeast Asia) and more ), South America (Brazil, Argentina, Colombia), the Middle East and Africa (Saudi Arabia, United Arab Emirates, Egypt, Nigeria and South Africa) and ROW.

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Table of Content

1 Introduction of Stem Cell Banking Storage Market1.1 Overview of the Market1.2 Scope of Report1.3 Assumptions

2 Executive Summary

3 Research Methodology3.1 Data Mining3.2 Validation3.3 Primary Interviews3.4 List of Data Sources

4 Stem Cell Banking Storage Market Outlook4.1 Overview4.2 Market Dynamics4.2.1 Drivers4.2.2 Restraints4.2.3 Opportunities4.3 Porters Five Force Model4.4 Value Chain Analysis

5 Stem Cell Banking Storage Market, By Deployment Model5.1 Overview

6 Stem Cell Banking Storage Market, By Solution6.1 Overview

7 Stem Cell Banking Storage Market, By Vertical7.1 Overview

8 Stem Cell Banking Storage Market, By Geography8.1 Overview8.2 North America8.2.1 U.S.8.2.2 Canada8.2.3 Mexico8.3 Europe8.3.1 Germany8.3.2 U.K.8.3.3 France8.3.4 Rest of Europe8.4 Asia Pacific8.4.1 China8.4.2 Japan8.4.3 India8.4.4 Rest of Asia Pacific8.5 Rest of the World8.5.1 Latin America8.5.2 Middle East

9 Stem Cell Banking Storage Market Competitive Landscape9.1 Overview9.2 Company Market Ranking9.3 Key Development Strategies

10 Company Profiles10.1.1 Overview10.1.2 Financial Performance10.1.3 Product Outlook10.1.4 Key Developments

11 Appendix11.1 Related Research

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Tags: Stem Cell Banking Storage Market Size, Stem Cell Banking Storage Market Growth, Stem Cell Banking Storage Market Forecast, Stem Cell Banking Storage Market Analysis, Stem Cell Banking Storage Market Trends, Stem Cell Banking Storage Market

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Stem Cell Banking Storage Market Size Analysis, Top Manufacturers, Shares, Growth Opportunities and Forecast to 2026 - Science In Me

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Canine Stem Cell Therapy Market 2020: Analysis By Regional Outlook, Competitive Landscape, Strategies And Forecasts 2026 – https://sciencein.me/

Canine Stem Cell Therapy Market (2018) Report Provides an in-depth summary of Canine Stem Cell Therapy Market Status as well as Product Specification, Technology Development, and Key Manufacturers. The Report Gives Detail Analysis on Market concern Like Canine Stem Cell Therapy Market share, CAGR Status, Market demand and up to date Market Trends with key Market segments.

The latest report about the Canine Stem Cell Therapy market provides a detailed evaluation of the business vertical in question, alongside a brief overview of the industry segments. An exceptionally workable estimation of the present industry scenario has been delivered in the study, and the Canine Stem Cell Therapy market size with regards to the revenue and volume have also been mentioned. In general, the research report is a compilation of key data with regards to the competitive landscape of this vertical and the multiple regions where the business has successfully established its position.

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Leading manufacturers of Canine Stem Cell Therapy Market:

Market Taxonomy

The global canine stem cell therapy market has been segmented into:

Product Type:

Application:

End User:

Region:

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Scope of The Canine Stem Cell Therapy Market Report:

This research report for Canine Stem Cell Therapy Market explores different topics such as product scope, product market by end users or application, product market by region, the market size for the specific product Type, sales and revenue by region forecast the Market size for various segments. The Report provides detailed information regarding the Major factors (drivers, restraints, opportunities, and challenges) influencing the growth of the Canine Stem Cell Therapy market. The Canine Stem Cell Therapy Market Report analyzes opportunities in the overall Canine Stem Cell Therapy market for stakeholders by identifying the high-growth segments.

A detailed overview of the geographical and competitive sphere of the Canine Stem Cell Therapy market:

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Table of Content of The Report

Chapter 1- Canine Stem Cell Therapy Industry Overview:

1.1 Definition of Canine Stem Cell Therapy

1.2 Brief Introduction of Major Classifications

1.3 Brief Introduction of Major Applications

1.4 Brief Introduction of Major Regions

Chapter 2- Production Market Analysis:

2.1 Global Production Market Analysis

2.1.1 Global Capacity, Production, Capacity Utilization Rate, Ex-Factory Price, Revenue, Cost, Gross and Gross Margin Analysis

2.1.2 Major Manufacturers Performance and Market Share

2.2 Regional Production Market Analysis

Chapter 3- Sales Market Analysis:

3.1 Global Sales Market Analysis

3.2 Regional Sales Market Analysis

Chapter 4- Consumption Market Analysis:

4.1 Global Consumption Market Analysis

4.2 Regional Consumption Market Analysis

Chapter 5- Production, Sales and Consumption Market Comparison Analysis

Chapter 6- Major Manufacturers Production and Sales Market Comparison Analysis

Chapter 7- Major Classification Analysis

Chapter 8- Major Application Analysis

Chapter 9- Industry Chain Analysis:

9.1 Up Stream Industries Analysis

9.2 Manufacturing Analysis

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Canine Stem Cell Therapy Market 2020: Analysis By Regional Outlook, Competitive Landscape, Strategies And Forecasts 2026 - https://sciencein.me/

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Young-onset Parkinson’s may start in the womb – Health24

People who develop Parkinson's disease at a younger age (before age 50) may have malfunctioning brain cells at birth, according to a study that also identified a drug that may help these patients.

At least 500 000 people in the United States are diagnosed with Parkinson's each year. Most are 60 or older at diagnosis, but about 10% are between 21 and 50.

Parkinson's is a neurological disease that occurs when brain neurons that make dopamine become impaired or die. Dopamine helps coordinate muscle movement.

Symptoms get worse over time and include slow gait, rigidity, tremors and loss of balance. There is currently no cure.

"Young-onset Parkinson's is especially heart-breaking because it strikes people at the prime of life," said study co-author Dr Michele Tagliati, director of the Movement Disorders Program at Cedars-Sinai Medical Center in Los Angeles.

"This exciting new research provides hope that one day we may be able to detect and take early action to prevent this disease in at-risk individuals," he said in a hospital news release.

For the study, Tagliati and colleagues generated special stem cells from the cells of patients with young-onset Parkinson's disease. These stem cells can produce any cell type of the human body. Researchers used them to produce dopamine neurons from each patient and analysed those neurons in the lab.

The dopamine neurons showed two key abnormalities: build-up of a protein called alpha-synuclein, which occurs in most forms of Parkinson's disease; and malfunctioning lysosomes, structures that act as "trash cans" for the cell to break down and dispose of proteins. This malfunction could result in a build-up of alpha-synuclein, the researchers said.

"Our technique gave us a window back in time to see how well the dopamine neurons might have functioned from the very start of a patient's life," said senior author Clive Svendsen, director of the Cedars Sinai Board of Governors Regenerative Medicine Institute.

"What we are seeing using this new model are the very first signs of young-onset Parkinson's," Svendsen said in the release. "It appears that dopamine neurons in these individuals may continue to mishandle alpha-synuclein over a period of 20 or 30 years, causing Parkinson's symptoms to emerge."

The study was published in the journal Nature Medicine.

The researchers also tested drugs that might reverse the neuron abnormalities. A drug called PEP005 already approved by the US Food and Drug Administration for treating pre-cancers of the skin reduced elevated levels of alpha-synuclein both in mice and in dopamine neurons in the lab.

The investigators plan to determine how PEP005, which is available in gel form, might be delivered to the brain to potentially treat or prevent young-onset Parkinson's.

They also want to find out whether the abnormalities in neurons of young-onset Parkinson's patients also exist in other forms of Parkinson's.

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Young-onset Parkinson's may start in the womb - Health24

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