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PubMed2006

Use of CFDA-SE for evaluating the in vitro proliferation pattern of human mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSC) are multipotent progenitors retaining the capability to undergo multilineage differentiation, mostly towards all the mesodermal cellular lineages. MSC growing under standard conditions are composed of two main subpopulations with a characteristic distribution in the morphologic flow cytometric scatter: RS (recycling stem) cells (small, agranular) and m (mature) MSC (large, moderately granular cells). METHODS: MSC obtained from BM of healthy donors and expanded in culture were characterized by evaluating both the expression of conventional markers and differentiation potential. We used CFSE, a lipophilic dye that is taken up by cell membranes, to investigate separately the proliferative activity of RS cells and mMSC subsets. RESULTS: With flow cytometric analysis, RS cells and mMSC showed nearly the same immunophenotypic pattern, even if a significantly smaller percentage of RS cells expressed some of the classic mesenchymal Ag. The RS cell fraction was confirmed to have a higher proliferative potential and such a feature was particularly evident under certain culture conditions. DISCUSSION: CFSE has been shown as a reliable method for studying the proliferative activity of MSC subpopulations identified by flow cytometric analysis. The acquisition parameter strategy is crucial for the accuracy of the analysis.

Adipocytes↗
PubMed1992

The consequences of a constitutive expression of MyoD1 in ES cells and mouse embryos.

A variety of differentiated cell types can be converted to skeletal muscle following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones tested, carrying single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 genes but not the endogenous MyoD1, MRF4, myf5, skeletal muscle actin or myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers was observed only when the transfected cells were allowed to differentiate, via embryoid bodies, in low mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and myf5 genes and in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of MyoD1 was not required for myogenesis. Interestingly, no preferential myogenesis was observed when the transfected ES cells were allowed to differentiate in vivo to teratocarcinomas. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function. Second, MyoD1 function in ES cells, even under environmental conditions that favour differentiation, is not dominant (incomplete penetrance). Third, that the exogenous MyoD1 transactivates the endogenous myogenin and MLC2 genes in ES cells. No live transgenic mice could be produced following microinjection of the beta-actin/MyoD1 gene into the pronuclei of fertilized eggs. Transgenic embryos died before mid gestation. The majority of tested embryos between 7.5 and 9.5 days, although retarded compared to control litermates, differentiated into tissues representative of all three germ layers. The expression of the introduced gene was detected in all ectodermal and mesodermal tissues but was absent in all endodermal cells. These results demonstrate again that MyoD1 is not a dominant regulatory factor.

Actins↗
PubMed1999

Neural stem cells -- a versatile tool for cell replacement and gene therapy in the central nervous system.

In recent years, it has become evident that the developing and even the adult mammalian central nervous system contains a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to a correct variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Animals↗
PubMed2001

[IDDM treatment with an insulin-expressing vector].

Islet transplantation has proven successful at delivering insulin in a regulated manner. However, the scarcities of islet donors are important limitations. The idea of engineering non-beta cell such as hepatocytes, myocytes, to produce insulin emerged at the end of 80's. To attain insulin production similarly to islet beta cell, a number of devices has been performed, including proinsulin processing and regulated secretion of insulin, resulting in functioning beta-cell surrogates to some extent. These attempts will be merged with currently on-going islet-regeneration therapies from multipotent embryonic stem (ES) cells.

Animals↗
PubMed2006

Neural differentiation of human mesenchymal stem cells: Evidence for expression of neural markers and eag K+ channel types.

OBJECTIVE: Mesenchymal stem cells (MSCs) are multipotent cells that can self-renew, proliferate, and exhibit elevated cellular plasticity. To investigate their possible neural fate, we studied human mesenchymal stem cells (hMSCs) in different cell culture conditions from morphological, immunochemical, gene expression, and physiological points of view. MATERIALS AND METHODS: We tested hMSCs in three previously reported experimental conditions made of alpha-modified minimum essential medium (alpha-MEM)/1 mM beta-mercaptoethanol (betaME), 10 microM alpha-MEM/retinoic acid (RA) or alpha-MEM/2% dimethylsulfoxide (DMSO) + 200 microM beta-hydroxyanisole (BHA), respectively, and in a new experimental condition with neural progenitor maintenance medium (NPMM). RESULTS: hMSCs were isolated from bone marrow and expanded for several passages. In betaME, cells became immunoreactive for neuronal nuclear antigen (NeuN), neuron-specific enolase (NSE), Nestin, and glial fibrillary acidic protein (GFAP). In experimental conditions with RA and DMSO/BHA, hMSCs were NeuN and NSE-positive while in NPMM they were positive for GFAP and NSE. Untreated hMSCs showed a weak mRNA expression for microtubule-associated protein, NSE, and neurofilament protein-medium and GFAP, which strongly increased in NPMM-treated hMSCs. In the electrophysiological study, NPMM-differentiated hMSCs expressed two delayed rectifier K+ currents related to two ether-à-go-go K+ channels (eag1, eag2), which are fundamental for setting the negative resting potentials required for neuronal survival and basal cell activity. The two K+ channels were absent in undifferentiated hMSCs. These data were confirmed by real-time polymerase chain reaction. CONCLUSION: In our new culture condition, hMSCs acquired new morphological characteristics, neural markers, and electrophysiological properties, which are suggestive of neural differentiation. This might lead to clinical use of hMSCs in neural degenerative diseases.

Cell Culture Techniques↗
PubMed2005

Properties of growth and molecular profiles of rat progenitor cells from ciliary epithelium.

Recent studies have demonstrated that multipotent retinal stem or progenitor cells can be isolated from the ciliary epithelium (CE) of the eye using a neurosphere culture. In this study, we investigated the properties of growth and differentiation, and molecular profiles of rat adult ciliary epithelium (CE)-derived retinal progenitors and forebrain (FB) derived neurospheres. Under clonogenic culture conditions, we found that the CE-derived neurospheres contained fewer undifferentiated cells compared with the FB-derived neurospheres, and that CE-derived neurospheres initially expressed the set of Notch pathway molecules genes including Notch 1 and Delta 1, HES-1 and HES-5, but partially lose their expression after passaging. Furthermore, we found that the CE-derived neurospheres did not express several markers for in vivo embryonic retinal progenitors. Additionally, when the eye was divided into four subregions along its dorsoventral and nasotemporal axes and progenitor cells were obtained from the subregions, the progenitor cells did not express the subregion specific transcription factors, suggesting that subregional specificity is not maintained in vitro. Together, our results demonstrate that CE-derived progenitor cells may have intrinsic limitations in the production of cell types.

Animals↗
PubMed2006

Gene expression profiling and analysis of signaling pathways involved in priming and differentiation of human neural stem cells.

Human neural stem cells have the ability to differentiate into all three major cell types in the CNS including neurons, astrocytes and oligodendrocytes. The multipotency of human neural stem cells shed a light on the possibility of using stem cells as a therapeutic tool for various neurological disorders including neurodegenerative diseases and neurotrauma that involve a loss of functional neurons. We have discovered previously a priming procedure to direct primarily cultured human neural stem cells to differentiate into almost pure neurons when grafted into adult CNS. However, the molecular mechanism underlying this phenomenon is still unknown. To unravel transcriptional changes of human neural stem cells upon priming, cDNA microarray was used to study temporal changes in human neural stem cell gene expression profile during priming and differentiation. As a result, transcriptional levels of 520 annotated genes were detected changed in at least at two time points during the priming process. In addition, transcription levels of more than 3000 hypothetical protein encoding genes and EST genes were modulated during the priming and differentiation processes of human neural stem cells. We further analyzed the named genes and grouped them into 14 functional categories. Of particular interest, key cell signal transduction pathways, including the G-protein-mediated signaling pathways (heterotrimeric and small monomeric GTPase pathways), the Wnt signaling pathway and the TGF-beta pathway, are modulated by the neural stem cell priming, suggesting important roles of these key signaling pathways in priming and differentiation of human neural stem cells.

Bone Morphogenetic Proteins↗
PubMed2002

Pluripotent stem cells--model of embryonic development, tool for gene targeting, and basis of cell therapy.

Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are derived from pre-implantation embryos and can be propagated as a homogeneous, uncommitted cell population for an almost unlimited period of time without losing their pluripotency and their stable karyotype. Murine ES cells are able to reintegrate fully into embryogenesis when returned into an early embryo, even after extensive genetic manipulation. In the resulting chimeric offspring produced by blastocyst injection or morula aggregation, ES cell descendants are represented among all cell types, including functional gametes. Therefore, mouse ES cells represent an important tool for genetic engineering, in particular via homologous recombination, to introduce gene knock-outs and other precise genomic modifications into the mouse germ line. Because of these properties ES cell technology is of high interest for other model organisms and for livestock species like cattle and pigs. However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have yet been established for vertebrate species other than the mouse (Evans and Kaufman, 1981; Martin, 1981) and chicken (Pain et al., 1996). The in vitro differentiation capacity of ES cells provides unique opportunities for experimental analysis of gene regulation and function during cell commitment and differentiation in early embryogenesis. Recently, pluripotent stem cells were established from human embryos (Thomson et al., 1998) and early fetuses (Shamblott et al., 1998), opening new scenarios both for research in human developmental biology and for medical applications, i.e. cell replacement strategies. At about the same time, research activities focused on characteristics and differentiation potential of somatic stem cells, unravelling an unexpected plasticity of these cell types. Somatic stem cells are found in differentiated tissues and can renew themselves in addition to generating the specialized cell types of the tissue from which they originate. Additional to discoveries of somatic stem cells in tissues that were previously not thought to contain these kinds of cells, they also appear to be capable of developing into cell types of other tissues, but have a reduced differentiation potential as compared to embryo-derived stem cells. Therefore, somatic stem cells are referred to as multipotent rather than pluripotent. This review summarizes characteristics of pluripotent stem cells in the mouse and in selected livestock species, explains their use for genetic engineering and basic research on embryonic development, and evaluates their potential for cell therapy as compared to somatic stem cells.

Animals↗
PubMed1999

Seeking muscle stem cells.

Skeletal muscle development requires the formation of myoblasts that can fuse with each other to form multinucleate myofibers. Distinct primary and secondary, slow and fast, populations of myofibers form by the time of birth. At embryonic, fetal, and perinatal stages of development, temporally distinct lineages of myogenic cells arise and contribute to the formation of these multiple types of myofibers. In addition, spatially distinct lineages of myogenic cells arise and form the anterior head muscles, limb (hypaxial) muscles, and dorsal (epaxial) muscles. There is strong evidence that myoblasts are produced from muscle stem cells, which are self-renewing cells that do not themselves terminally differentiate but produce progeny that are capable of becoming myoblasts and myofibers. Muscle stem cells, which may be multipotent, appear to be distinguishable from myoblasts by a number of indirect and direct criteria. Muscle stem cells arise either in unsegmented paraxial mesoderm (anterior head muscle progenitors) or in segmented mesoderm of the somites (epaxial and hypaxial muscle progenitors). These initial stages of myogenesis are regulated by positive and negative signals, including Wnt, BMP, and Shh family members, from nearby notochord, neural tube, ectoderm, and lateral mesoderm tissues. The formation of skeletal muscles, therefore, depends on the generation of spatially and temporally distinct lineages of myogenic cells. Myogenic cell lineages begin with muscle stem cells which produce the myoblasts that fuse to form myofibers.

Animals↗
PubMed2005

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells.

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric alpha-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty.

Adipocytes↗
PubMed2006

Epigenetics and the plasticity of differentiation in normal and cancer stem cells.

Embryonic stem cells are characterized by their differentiation to all cell types during embryogenesis. In adult life, different tissues also have somatic stem cells, called adult stem cells, which in specific niches can undergo multipotent differentiation. The use of these adult stem cells has considerable therapeutic potential for the regeneration of damaged tissues. In both embryonic and adult stem cells, differentiation is controlled by epigenetic mechanisms, and the plasticity of differentiation in these cells is associated with transcription accessibility for genes expressed in different normal tissues. Abnormalities in genetic and/or epigenetic controls can lead to development of cancer, which is maintained by self-renewing cancer stem cells. Although the genetic abnormalities produce defects in growth and differentiation in cancer stem cells, these cells have not always lost the ability to undergo differentiation through epigenetic changes that by-pass the genomic abnormalities, thus creating the basis for differentiation therapy. Like normal stem cells, cancer stem cells can show plasticity for differentiation. This plasticity of cancer stem cells is also associated with transcription accessibility for genes that are normally expressed in different tissues, including tissues other than those from which the cancers originated. This broad transcription accessibility can also contribute to the behavior of cancer cells by overexpressing genes that promote cell viability, growth and metastasis.

Adult Stem Cells↗
PubMed1997

Patterns of organ-specific engraftment by stem cell subsets and committed progenitors.

The kinetics of blood and organ engraftment following transplants of defined populations of hematopoietic stem/progenitor cells were investigated utilizing cell populations defined by surface antigen and rhodamine-123 staining. While long-term repopulating stem cells, short-term multipotent progenitors and committed progenitors all reconstituted peripheral blood red cells and splenic cellularity, only the population of cells that includes highly enriched long-term repopulating stem cells (Thy-1.1lowLinnegSca-1+Rh123low) reconstituted marrow cellularity. In addition, peripheral blood platelet and nucleated cell count increased only after transplant of the long-term repopulating population. These results argue that the major cell population that functions to reconstitute hematopoiesis after bone marrow transplantation is a primitive, marrow-homing stem cell. Transplantation of highly enriched multipotent progenitors that lack long-term reconstituting potential had no impact on hematopoietic recovery, apart from a transient increase in circulating erythrocytes. These results suggest that the primary cell population that functions to reconstitute hematopoiesis in a transplant setting is the long-term repopulating stem cell. This observation is discussed in the context of the normal hematopoietic process.

Animals↗
PubMedDate not supplied

Adult neural stem cells: plasticity and developmental potential.

Stem cells play an essential role during the processes of embryonic tissue formation and development and in the maintenance of tissue integrity and renewal throughout adulthood. The differentiation potential of stem cells in adult tissues has been thought to be limited to cell lineages present in the organ from which they derive, but there is evidence that somatic stem cells may display a broader differentiation repertoire. This has been documented for bone marrow stem cells (which can give rise to muscle, hepatic and brain cells) and for muscle precursors, which can turn into blood cells. The adult central nervous system (CNS) has long been considered incapable of cell renewal and structural remodeling. Recent findings indicate that, even in postnatal and adult mammals, neurogenesis does occur in different brain regions and that these regions actually contain adult stem cells. These cells can be expanded both in vivo and ex vivo by exposure to different combinations of growth factors and subsequently give rise to a differentiated progeny comprising the major cell types of the CNS. Almost paradoxically, adult neural stem cells display a multipotency much broader than expected, since they can differentiate into non-CNS mesodermal-derivatives, such as blood cells and skeletal muscle cells. We review the recent findings documenting this unforeseen plasticity and unexpected developmental potential of somatic stem cells in general and of neural stem cells in particular. To better introduce these concepts, some basic notions on the functional properties of adult neural stem cells will also be discussed, particularly focusing on the emerging role of the microenvironment in determining and maintaining their peculiar characteristics.

Aging↗
PubMed1995

Impaired cytokine production by bone marrow stromal cells of immunodeficient mice.

The aetiology of the bone marrow suppression in HIV-infected patients is unknown. We have demonstrated previously that the ability of bone marrow cells, derived from mice made immunodeficient by infection with the retrovirus LP-BM5, to establish long-term stromal cultures is impaired. In this study we determined the ability of bone marrow stromal cells from these immunodeficient mice to produce cytokines important in haemopoiesis. Neither SCF, IL-3, GM-CSF nor TNF alpha were found in conditioned media of long-term bone marrow cultures (LTBMC) of normal or MAIDS mice. We failed to detect mRNA for TNF or IL-1 alpha, by reverse transcription polymerase chain reaction (RT-PCR), in cultures derived from either normal or immunodeficient mice. Steady-state levels of transcripts for IL-6 were equal in cells from normal and MAIDS mice. Steady-state levels of mRNA for TGF beta 1, a known inhibitor of haemopoiesis, were decreased in cultures derived from MAIDS mice at late stages of infection. The mRNA level of the multipotent haemopoietic regulator stem cell factor was also decreased in MAIDS cultures as compared with normals. Transcripts encoding the transmembrane form of the growth factor were almost absent. Addition of soluble GM-CSF and SCF only transiently increased the production of CFUs (BFUE and CFU-G/M) in MAIDS LTBMC. These findings suggest that derangements in cytokine production in stromal cells of immunodeficient mice may contribute to the suppression of haemopoiesis observed in this disease. One mechanism of HIV-induced depression of haemopoiesis may be via alterations of the haemopoietic microenvironment.

Animals↗
PubMed1985

The hematopoietic stem cells of alpha-thalassemic mice.

The alpha-thalassemic mouse has a hereditary microcytic anemia, almost certainly has a shortened RBC life span, and is a potential candidate for cell replacement therapy. In a routine study of bone marrow repopulating capacity using hemoglobin as a cell marker, normal donor marrow cells, but not alpha-thalassemic donor marrow cells, completely replaced the host cells. Further analysis showed that at least 30 times more alpha-thalassemic cells were required to outcompete normal donor cells injected simultaneously. The results were more extreme then expected and suggested a defect in a stem cell population as well as in the RBCs. Evidence that the multipotent and erythroid-committed stem cells in alpha-thalassemic mice are not decreased was shown by CFU-S and CFU-E assays. The combined results indicate that the deletion expresses itself most conspicuously in the RBC population. Tests were also performed to analyze repopulation kinetics in the Hbath-J/+ mice. In unirradiated alpha-thalassemic hosts, the hemoglobin from a normal donor persisted but did not replace the host hemoglobin. Sublethally irradiated alpha-thalassemic hosts, on the other hand, were easily repopulated with normal cells. We conclude that the alpha-thalassemic mouse is a good model for cell replacement therapy.

Animals↗
PubMed2000

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase.

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.

Adipose Tissue↗
PubMed1986

Differentiation alters the unstable expression of adenine phosphoribosyltransferase in mouse teratocarcinoma cells.

Three multipotent mouse teratocarcinoma stem lines, all exhibiting unstable expression for the purine salvage enzyme adenine phosphoribosyltransferase (APRT) were used for the isolation of differentiated cell lines from neoplasms developed in syngeneic mice. Two of the stem cell lines (DAP1B and DAP1C) exhibited homozygous deficiencies for APRT expression while the third stem cell line (E140) exhibited a heterozygous deficiency (Turker, M.S., Smith, A.C., and Martin, G.M.; Somat. Cell Mol. Genet.; 10:55-69; 1984). A total of 16 morphologically differentiated cell lines were established from these neoplasms; most were no longer tumorigenic. Differentiated cell lines derived from the E140-induced tumors segregated homozygous deficient mutants in a single step, consistent with their retention of the heterozygous deficient state. Differentiated homozygous deficient cell lines gave rise to phenotypic revertants at very high frequencies (10(-1) to 10(-2)). The majority of these putative revertants, however, yielded cell-free extracts with little or no detectable APRT activity. These putative revertants were capable of adenine salvage and were therefore termed APRT pseudorevertants. Since the APRT pseudorevertant phenotype was only observed in the differentiated progeny of the APRT deficient stem cell lines, we conclude that this change in the nature of the revertant phenotype was a consequence of cellular differentiation.

Adenine↗
PubMed2005

Proliferative activity of adipocytes in adipose tissue tumors.

Electron-autoradiographic study of normal and tumor-transformed adipose tissue (common lipoma and destructive lipoma, i.e. infiltrating and degrading lipoma) showed the capacity of adipose tissue cells in lipomas, especially in destructive lipomas, to proliferation and differentiation. In vivo synthesis of DNA in mature adipocytes not observed previously is described. The role of microvascular wall cells as mesenchymal multipotent precursors in the formation of the adipose tissue is discussed. The involvement of the bone marrow mesenchymal stem cell in this process cannot be ruled out.

Adipocytes↗