Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone

RCJ 3.1, a clonally derived cell population isolated from 21-d fetal rat calvaria, expresses the osteoblast-associated characteristics of polygonal morphology, a cAMP response to parathyroid hormone, synthesis of predominantly type I collagen, and the presence of 1,25-dihydroxyvitamin D3-regulated alkaline phosphatase activity. When cultured in the presence of ascorbic acid, sodium beta-glycerophosphate, and the synthetic glucocorticoid dexamethasone, this clone differentiated in a time-dependent manner into four morphologically distinct phenotypes of known mesenchymal origin. Multinucleated muscle cells were observed as early as 9-10 d in culture, lipid-containing adipocytes formed after 12 d, chondrocyte nodules were observed after 16 d, and mineralized bone nodules formed after 21 d in culture. The differentiated cell types were characterized morphologically, histochemically, and immunohistochemically. The formation of adipocytes and chondrocytes was dependent upon the addition of dexamethasone; the muscle and bone phenotypes were also expressed at low frequency in the absence of dexamethasone. The sex steroid hormones progesterone and 17 beta-estradiol had no effect on differentiation in this system, suggesting that the effects of dexamethasone represent effects specific for glucocorticosteroids. Increasing concentrations of dexamethasone (10(-9)-10(-6) M) increased the numbers of myotubes, adipocytes, and chondrocytes; however, when present continuously for 35 d, the lower concentrations appeared to better maintain the muscle and adipocyte phenotypes. Bone nodules were not quantitated because the frequency of bone nodule formation was too low. Single cells obtained by plating RCJ 3.1 cells at limiting dilutions in the presence of dexamethasone, were shown to give rise to subclones that could differentiate into either single or multiple phenotypes. Thus, the data suggest that this clonal cell line contains subpopulations of mesenchymal progenitor cells which can, under the influence of glucocorticoid hormones, differentiate in vitro into four distinct cell types. It is, therefore, a unique cell line which will be of great use in the study of the regulation of mesenchymal stem cell differentiation.     

Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes

The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or "biomarkers," of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations.     

Mesodermal cell lineage determination and proto-oncogene expression

Previously, we have established a rat embryo fibroblastic cell line that is characterized as immortal, nontumorigenic and retains a diploid karyotype. We report here that this cell line will spontaneously differentiate along mesodermal cell lineages to form myotubes and adipocytes. We have isolated and characterized lineage-determined (nondifferentiated) preadipocytes and myoblasts. Using these cell lines we have investigated the expression of proto-oncogenes concomitant with lineage-specific determination. No major changes in proto-oncogene RNA levels were observed that correlated with lineage determination. These results would suggest that none of the 15 proto-oncogenes used in these experiments are involved in mesodermal lineage determination; but this does not rule out the possibility that other proto-oncogenes not tested or currently unknown may be involved. However, these differentiating subclones and nondifferentiating fibroblasts that exhibit a normal karyotype will be an excellent model-system to investigate the molecular basis of cell lineage determination.     

Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.

Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T1/2CL8 mouse cells treated with 5-azacytidine or 5-aza-2'-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T1/2CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T1/2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.     

Cell diversification within the myogenic lineage: in vitro generation of two types of myoblasts from a single myogenic progenitor cell

We show that a single myogenic progenitor cell in vitro generates two types of myoblasts committed to two distinct myogenic cell lineages. Using fast and slow myosin heavy chain isoform content to define myotube type, we found that myogenic cells from fetal quail (day 10 in ovo) formed two types of myotubes in vitro: fast and mixed fast/slow. Clonal analysis showed that these two types of myotubes were formed from two types of myoblasts committed to distinct fast and fast/slow lineages. Serial subcloning demonstrated that the initial myoblast progeny of an individual myogenic progenitor cell were in the fast lineage, whereas later progeny were in the fast/slow lineage. Fast and slow myosin expression within particular myotubes reflects the genetic processes underlying myoblast commitment to diverse myogenic lineages.     

Do microRNAs regulate bone marrow stem cell niche physiology?

The adult bone marrow, situated within the bone cavity, comprises three distinct stem cell populations: hematopoietic stem cells (HSCs), mesenchymal stromal/stem cells (MSCs) and endothelial progenitor/stem cells (EPCs). HSCs are a well-characterized population of self-renewing cells that give rise to all blood cells. The definition of MSCs is more complex due to the limited understanding of MSC properties. In general, MSCs are considered multipotent stromal cells that are able to differentiate into various cell types, including osteoblasts, chondrocytes and adipocytes. Compared to HSCs and MSCs, EPCs are a newly discovered population of stem/progenitor cells with the capacity to differentiate into endothelial cells, the cells forming the inner lining of a blood vessel.     

A versatile chondrogenic rat calvaria cell line R-tTA-24 that permits tetracycline-regulated gene expression

The clonal rat calvaria cell line RCJ3.1C5.18 (RCJ) undergoes chondrogenic differentiation after long-term culture post confluence. To allow flexible genetic manipulation, a tetracycline-regulated gene expression system was established in this cell line. Treatment with tetracycline in operational doses does not affect the differentiation of RCJ cells with respect to the markers tested. After stable transfection with pUHD15.1 containing the tetracycline transactivator (tTA) in the presence of pTK-hyg for hygromycin selection, 28 clones were isolated and characterized for alcian blue staining of cartilage-specific proteoglycans and for collagen type II expression. Clone R-tTA-24 was selected on the basis of phenotype and displayed tetracycline-dependent downregulation of luciferase activity (tet-OFF system) by two orders of magnitude (57–149-fold) after stable transfection with the reporter gene pBI-EGFP/luc. The novel, chondrogenic cell line R-tTA-24 may be stably transfected with various genes of interest for tetracycline- regulated gene expression using neomycin selection and may be a valuable tool to study the process of chondrogenic differentiation in vitro. Accepted: 30 November 1999     

Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells

Mesenchymal stem cells (MSC) are considered as potential agents for reconstructive and gene-targeting therapies since they differentiate into various cell-lineages, exhibit an extended survival once injected into a host, and can easily be transfected with engineered DNA. MSC are essentially isolated from hematopoietic bone marrow (BM), a process that is rather invasive and may raise ethical concerns. In an attempt to find an alternative source, we evaluated whether non-hematopoietic (nh)BM recovered from femoral heads of patients undergoing hip arthroplasty contained MSC. Ex vivo, 99% of nhBM cells were CD45(+) leukocytes. After culture, leukocytes were replaced by a homogeneous layer of adherent CD45(-) CD14(-) CD34(-) CD11b(-) CD90(+) HLA-ABC(+) cells. Culture doubling time (mean = 4 days, range 1.6-6.7 days) was not correlated with patient age (27-81 years, n = 16). Amplified cultures supported long-term hematopoiesis, and could be differentiated in vitro into adipocytes and chondrocytes. Moreover, a small fraction of nhBM cells spontaneously expressed MyoD1 and formed myotubes, suggesting that myogenic differentiation also occurred. nhBM contained clonogenic cells whose frequency (1/13,000), doubling time (2.1 days), and maximal amplification (up to 10(6)-fold) were not age-related. All 14 clones analyzed (from five patients, ages 27-78 years) differentiated into at least one mesenchymal lineage, and 66% were bipotential (n = 8/12), or tripotential (n = 2/3). In conclusion, nhBM contains pluripotential mesenchymal progenitors which are similar to hematopoietic BM-derived MSC, and whose biological functions are not altered by aging. Furthermore, if MSC-based therapies hold their promises, nhBM may become the source of choice for responding to the increasing demand for MSC.     

All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells

While it is clear that a single hematopoietic stem cell?(HSC) is capable of giving rise to all other hematopoietic cell types, the differentiation paths beyond HSC remain controversial. Contradictory reports on?the lineage potential of progenitor populations have questioned their physiological contribution of progenitor populations to multilineage differentiation. Here, we established a lineage tracing mouse model that enabled direct assessment of differentiation pathways in?vivo. We provide definitive evidence that differentiation into all hematopoietic lineages, including megakaryocyte/erythroid cell types, involves Flk2-expressing non-self-renewing progenitors. A Flk2+ stage was used during steady-state hematopoiesis, after irradiation-induced stress and upon HSC transplantation. In contrast, HSC origin and maintenance do not include a Flk2+ stage. These data demonstrate that HSC specification and maintenance are Flk2 independent, and that hematopoietic lineage separation occurs downstream of Flk2 upregulation.     

Clonal analysis of the differentiation potential of human adipose-derived adult stem cells

Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.     

In vitro differentiation of mesenchymal progenitor cells derived from porcine umbilical cord blood

Mesenchymal stem/progenitor cells (MPCs) were isolated from porcine umbilical cord blood (UCB) and their morphology, proliferation, cell cycle status, cell-surface antigen profile and expression of hematopoietic cytokines were characterized. Their capacity to differentiate in vitro into osteocytes, adipocytes and chondrocytes was also evaluated. Primary cultures of adherent porcine MPCs (pMPCs) exhibited a typical fibroblast-like morphology with significant renewal capacity and proliferative ability. Subsequent robust cell growth was indicated by the high percentage of quiescent (G0/G1) cells. The cells expressed the mesenchymal surface markers, CD29, CD49b and CD105, but not the hematopoietic markers, CD45 and CD133 and synthesized hematopoietic cytokines. Over 21 days of induction, the cells differentiated into osteocytes adipocytes and chondrocytes. The expression of lineage specific genes was gradually upregulated during osteogenesis, adipogenesis and chondrogenesis. We conclude that porcine umbilical cord blood contains a population of MPCs capable of self-renewal and of differentiating in vitro into three classical mesenchymal lineages.     

Development and regeneration in the central nervous system

As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.     

Neurogenin 3 and the enteroendocrine cell lineage in the adult mouse small intestinal epithelium

It is thought that small intestinal epithelial stem cell progeny, via Notch signaling, yield a Hes1-expressing columnar lineage progenitor and an Atoh1 (also known as Math1)-expressing common progenitor for all granulocytic lineages including enteroendocrine cells, one of the body's largest populations of endocrine cells. Because Neurogenin 3 (Neurog3) null mice lack enteroendocrine cells, Neurog3-expressing progenitors derived from the common granulocytic progenitor are thought to produce the enteroendocrine lineage, although more recent work indicates that Neurog3+ progenitors also contribute to non-enteroendocrine lineages. We aimed to test this model and better characterize the progenitors leading from the stem cells to the enteroendocrine lineage. We investigated clones derived from enteroendocrine precursors and found no evidence of a common granulocytic progenitor that routinely yields all granulocytic lineages. Rather, enteroendocrine cells are derived from a short-lived bipotential progenitor whose offspring, probably via Notch signaling, yield a Neurog3+ cell committed to the enteroendocrine lineage and a progenitor committed to the columnar lineage. The Neurog3+ cell population is heterogeneous; only about 1/3 are slowly cycling progenitors, the rest are postmitotic cells in early stages of enteroendocrine differentiation. No evidence was found that Neurog3+ cells contribute to non-enteroendocrine lineages. Revised lineage models for the small intestinal epithelium are introduced.     

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.     

Simultaneous isolation of human BM hematopoietic, endothelial and mesenchymal progenitor cells by flow sorting based on aldehyde dehydrogenase activity: implications for cell therapy

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br), and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations), to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD, n=30) nucleated cells and was enriched in cells expressing CD34, CD117, CD105, CD127, CD133 and CD166, and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells, and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes, osteoblasts and chondrocytes. DISCUSSION: Hematopoietic, endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications.     

In vitro and in vivo properties of distinct populations of amniotic fluid mesenchymal progenitor cells

Human mesenchymal progenitor cells (MPCs) are considered to be of great promise for use in tissue repair and regenerative medicine. MPCs represent multipotent adherent cells, able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes or chondrocytes. Recently, we identified and characterized human second trimester amniotic fluid (AF) as a novel source of MPCs. Herein, we found that early colonies of AF-MPCs consisted of two morphologically distinct adherent cell types, termed as spindle-shaped (SS) and round-shaped (RS). A detailed analysis of these two populations showed that SS-AF-MPCs expressed CD90 antigen in a higher level and exhibited a greater proliferation and differentiation potential. To characterize better the molecular identity of these two populations, we have generated a comparative proteomic map of SS-AF-MPCs and RS-AF-MPCs, identifying 25 differentially expressed proteins and 10 proteins uniquely expressed in RS-AF-MPCs. Furthermore, SS-AF-MPCs exhibited significantly higher migration ability on extracellular matrices, such as fibronectin and laminin in vitro, compared to RS-AF-MPCs and thus we further evaluated SS-AF-MPCs for potential use as therapeutic tools in vivo. Therefore, we tested whether GFP-lentiviral transduced SS-AF-MPCs retained their stem cell identity, proliferation and differentiation potential. GFP-SS-AF-MPCs were then successfully delivered into immunosuppressed mice, distributed in different tissues and survived longterm in vivo. In summary, these results demonstrated that AF-MPCs consisted of at least two different MPC populations. In addition, SS-AF-MPCs, isolated based on their colony morphology and CD90 expression, represented the only MPC population that can be expanded easily in culture and used as an efficient tool for future in vivo therapeutic applications.     

Telomerase deficiency impairs differentiation of mesenchymal stem cells

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR(-/-)) and wild-type (WT) mice. The MSCs isolated from mTR(-/-) mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR(-/-)MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.     

The complex relationship between cell division and the control of differentiation in oligodendrocyte-type-2 astrocyte progenitor cells isolated from perinatal and adult rat optic nerves

By studying the response of a well-defined progenitor cell to two well-defined mitogens, we have been able to provide a dramatic example of the complex relationship which can exist between the control of cell division and the control of differentiation.In previous studies we have described the development of the oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell, a glial progenitor cell isolated from the rat optic nerve. Although originally described as a bipotential cell, we have recently identified a new differentiation pathway in this lineage. We have found that O-2A^perinatal progenitors, with properties appropriate for early development, give rise to O-2A^adult progenitors, which have stem cell-like properties more appropriate to the physiological needs of adult animals. Our studies thus indicate that the population of O-2A^perinatal progenitors is tripotential, and also suggests a possible developmental origin for self-renewing stem cells. Moreover, the properties of O-2A^adult progenitor cells may provide a cellular biological basis for understanding the failure of remyelination in multiple sclerosis.The division of both O-2A^perinatal and O-2A^adult progenitors is stimulated by type-1 astrocytes (which are themselves derived from a separate glial lineage) but this cell-cell interaction promotes different programs of differentiation in the two progenitor populations. The effects of type-1 astrocytes on perinatal and adult progenitors appears to be mediated by platelet-derived growth factor (PDGF), and this mitogen will also induce different programs of differentiation in the two progenitor populations. Moreover, the patterns of differentiation promoted by PDGF are different from those promoted by fibroblast growth factor (FGF), demonstrating that the modulation of division can be distinguished from the modulation of differentiation.     

5-azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: Evidence for regulatory genes controlling determination

5-Azacytidine converts the mouse embryonic cell line C3H10T1/2 into differentiated chondrocytes, adipocytes, and skeletal muscle. Clonal and 2D protein gel analyses demonstrate that 5-azacytidine converts 10T1/2 cells into three stably determined, but undifferentiated, stem cell lineages which can differentiate into myofibers, chondrocytes, and adipocytes. Conversion of 10T1/2 cells is accompanied by specific changes in protein synthetic patterns unique for each cell lineage. We propose that 5-azacytidine converts 10T1/2 cells by hypomethylation of “determination” regulatory loci which establish lineages of stem cells with a restricted potential to differentiate into muscle, cartilage, or fat cells. Our results suggest that these three lineages are specified by separate regulatory loci and that as few as 1–3 hypomethylation events per cell are sufficient to activate the hypothesized muscle regulatory locus. Conversion of 10T1/2 cells by 5-azacytidine provides a model for studying regulatory genes involved in cell lineage determination.