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PubMed2006

Mesenchymal lineage potentials of aorta-gonad-mesonephros stromal clones.

BACKGROUND AND OBJECTIVES: The characterization of stem cell microenvironments throughout ontogeny is of fundamental interest in the field of stem cell biology. Within the adult blood system, hematopoietic stem cells (HSC) are supported in the osteoblastic and endothelial bone marrow microenvironments. During mouse mid-gestation, the first HSC emerge autonomously in the aorta-gonad-mesonephros (AGM) region. However, little is known about this microenvironment. To study the cellular complexity of the AGM hematopoietic microenvironment and its relationship to HSC, we examined the potential of AGM stromal clones to differentiate into several mesenchymal lineages. DESIGN AND METHODS: Stromal cell clones from the mid-gestation mouse were cultured in appropriate conditions known to support osteogenic, adipogenic, chondrogenic and endothelial differentiation. Potentials of the stromal cells were scored by morphological examination of the cultures, specific staining and gene expression profile. RESULTS: We show that most clones possess uni/bilineage osteogenic, adipogenic and/or endothelial potential. The differentiation potential of the stromal clones appears to relate to their site of origin but not to their ability to support hematopoiesis. Moreover, we show that AGM HSC activity is unaffected by the osteogenic differentiation of UG26.1B6 stromal cells. INTERPRETATION AND CONCLUSIONS: These results confirm the existence of mesenchymal stem/progenitor cells in the AGM region and suggest that the AGM hematopoietic microenvironment is highly complex, containing stromal cells with various mesenchymal lineage potentials.

Animals↗
PubMed2006

Differentiation versus plasticity: fixing the fate of undetermined adult stem cells.

Adult mesenchymal stem cells own a considerable plasticity, which enables them to respond to various extra- and intracellular cues and exert, at least partially, various differentiation pathways. Recently, we have shown that multipotent adult stem cells (MASCs) derived from the mouse bone marrow (mBM-MASCs) consist of distinct cell populations that have similar differentiation abilities but differ in the expression of several stem cell markers. Despite their remarkable developmental potential MASCs seem to miss crucial components to complete specific differentiation programs necessary to acquire a fully functional phenotype. A thorough analysis of active and inactive regulatory circuits in multipotent adult stem cells might lead the way to a controlled programming of these cells and rational therapeutic applications.

Adult Stem Cells↗
PubMed2004

Human adult craniofacial muscle-derived cells: neural-cell adhesion-molecule (NCAM; CD56)-expressing cells appear to contain multipotential stem cells.

Skeletal muscle has been well characterized as a reservoir of myogenic precursors or satellite cells with the potential to participate in cellular repopulation therapies for muscle dysfunction. Recent evidence, however, suggests that the postnatal muscle compartment can be considered an alternative to bone marrow as a source of multipotent cells or muscle-derived stem cells (MDSCs). MDSCs, when primed with appropriate environmental cues, can differentiate into a variety of non-muscle cells. The present study describes the application of a new technique for the isolation of adult human myoblasts and putative MDSCs, based on microbead-immunomagnetic selection of CD56+ cells, derived from craniofacial skeletal muscle, and details changes in morphological/molecular phenotype of the purified cells when maintained in either a myogenic or a non-myogenic milieu. Multiple immunofluorescence microscopy and two-colour flow-cytometric analysis of proliferating CD56+ cultures revealed positive staining for myogenic markers (CD56, desmin and M-cadherin) as well as putative stem-cell markers [the antigens CD34, CD90 and CD106, and Flk-1 (fetal liver kinase-1)/VEGFR-2 (vascular-endothelial-growth-factor receptor)]. Confluent cultures subjected to cycles of adipogenic or osteogenic induction contained either adipocytes or osteoblasts and myotubes. In conclusion, the CD56+ subpopulation within adult human skeletal muscle is heterogeneous and is composed of both lineage-committed myogenic cells and multipotent cells (the candidate MDSCs), which are able to form non-muscle tissue such as fat and bone.

Adipocytes↗
PubMed2004

How important are adult stem cells for tissue maintenance?

Most tissues undergo significant expansion during postnatal life, as well as a lifelong cellular turnover to compensate for cell loss. What is the identity of cells that give rise to newly differentiated cells? This fundamental yet understudied question is now attracting the attention of biologists and clinicians, triggered by reports that essentially every tissue can be replenished and repaired by multipotent adult stem cells. The identification of the "cell of origin" for a given tissue is essential for understanding its dynamics during adult life, and may have important therapeutic implications for both degenerative and neoplastic diseases. In this commentary, we briefly outline classic and current views on the question of the cell of origin. We also describe a general method that we have recently developed for addressing this issue, and its first application for the study of pancreatic beta cells.

Animals↗
PubMedDate not supplied

Ovine cord blood accommodates multipotent mesenchymal progenitor cells.

BACKGROUND: Stem cells derived from umbilical cord blood display mesenchymal multipotency and can differentiate into osteoblasts, chondroblasts and adipoblasts in vitro under defined stimuli. Although sheep have been used as experimental models for investigations on xenoreactivity after transplantation of stem cells isolated from human umbilical cord blood, the potential of ovine cord blood stem cells to differentiate has been examined to date. MATERIALS AND METHODS: Mononuclear cells from the placentoms of 3 lambs were isolated via density gradient centrifugation and cultivated. After expansion up to 3 passages, the cells were stimulated to differentiate towards osteogenic (dexamethasone, ascorbic-acid-2-phosphate, beta-glycerolphosphate), chondrogenic (TGF-beta3, insulin, transferrin, selenium, dexamethasone, ascorbic-acid-2-phosphate) and adipogenic (indomethacine, insulin, 3-isobutyl-1-methylxanthine, dexamethasone) lines for 20 days. The cells were characterized morphologically by transmission and phase contrast light microscopy during lineage-specific stimulation. Immunocytochemistry and conventional stains were used to detect lineage-typical markers: fat vacuoles and peroxisome proliferation-acitivated receptor gamma2 (PPAR) served to detect adipoblasts, whereas osteopontin (OP) was used to characterize osteoblasts. A positive antibody reaction to collagen II and chondrogenic oligomeric protein (COMP) revealed the presence of chondroblasts. RESULTS: The osteogenic line formed bone nodules, adipogenic cells developed lipid droplets and the cells of the chondrogenic line showed typical chondroblast-like morphology. CONCLUSION: It was demonstrated that ovine mesenchymal stem cells, derived from umbilical cord blood (sheep unrestricted somatic stem cells, S-USSCs), can be isolated via gradient density centrifugation and expanded in vitro. Under lineage-specific stimulation, S-USSCs differentiated into osteo-, chondro- and adipoblasts with typical morphological characteristics. Significant quantitative differences between the stimulated and control groups in lineage-typical immunocytochemical markers verified these findings.

Adipose Tissue↗
PubMed2003

Parvovirus infection suppresses long-term repopulating hematopoietic stem cells.

The functional disturbance of self-renewing and multipotent hematopoietic stem cells (HSCs) in viral diseases is poorly understood. In this report, we have assessed the susceptibility of mouse HSCs to strain i of the autonomous parvovirus minute virus of mice (MVMi) in vitro and during persistent infection of an immunodeficient host. Purified 5FU(r) Lin(-) Sca-1(+) primitive hematopoietic precursors were permissive for MVMi genome replication and the expression of viral gene products. The lymphoid and myeloid repopulating capacity of bone marrow (BM) cells was significantly impaired after in vitro infection, although the degree of functional effect proportionally decreased with the posttransplantation time. This indicated that MVMi targets the heterogeneous compartment of repopulating cells with differential affinity and suggests that the virus may persist in some primitive HSCs in the quiescent stage, killing those eventually recruited for proliferative activity. Immunodeficient SCID mice oronasally infected with MVMi were cured of the characteristic virus-induced lethal leukopenia by transplantation of immunocompetent BM grafts. However, two double-stranded viral DNA species, probably uncommon replicative intermediates, remained in the marrow of every transplanted mouse months after infectious virus clearance. Genetic analysis of the rescued mice showed that the infection ensured a stable engraftment of donor hematopoiesis by markedly depleting the pool of endogenous HSCs. The MVMi-induced suppression of HSC functions illustrates the accessibility of this compartment to infection during a natural viral hematological disease. These results may provide clues to understanding delayed hematopoietic syndromes associated with persistent viral infections and to prospective gene delivery to HSCs in vivo.

Animals↗
PubMed2006

Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein.

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-gamma2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.

Adenoviridae↗
PubMed2007

Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix.

Adipose-derived stem cells (ASCs) are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo. Osterix (Osx) is a zinc finger-containing transcription factor of Sp gene family, which plays important roles in bone development and mineralization. In this study, we hypothesized that overexpression of Osx in murine ASCs would promote their osteogenic differentiation in vitro. A plasmid expressing Osx (pcDNA3.1-Osx) was constructed and applied to transfect monolayers of murine ASCs. Then expression of bone-related genes, nodule formation, proliferation rate, and alkaline phosphatase activity were examined to evaluate the osteogenic potential of ASCs with pcDNA3.1-Osx transfection. Results of RT-PCR and immunohistochemistry showed that pcDNA3.1-Osx transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, osteopontin, and Collagen type I in ASCs. At the same time, overexpression of Osx in ASCs enhanced alkaline phosphatase activity and capability to form mineralized nodules, while not inhibited their proliferation rate. These results indicated that pcDNA3.1-Osx transfection promoted the osteogenic differentiation of ASCs, while not affecting their proliferative ability. Since they can be easily isolated and genetically modified, ASCs are hopeful cell sources in the further application of hard tissue engineering.

Adipose Tissue↗
PubMed2004

Functional expression of HGF and HGF receptor/c-met in adult human mesenchymal stem cells suggests a role in cell mobilization, tissue repair, and wound healing.

Human mesenchymal stem cells (hMSC) are adult stem cells with multipotent capacities. The ability of mesenchymal stem cells to differentiate into many cell types, as well as their high ex vivo expansion potential, makes these cells an attractive therapeutic tool for cell transplantation and tissue engineering. hMSC are thought to contribute to tissue regeneration, but the signals governing their mobilization, diapedesis into the bloodstream, and migration into the target tissue are largely unknown. Here we report that hepatocyte growth factor (HGF) and the cognate receptor HGFR/c-met are expressed in hMSC, on both the RNA and the protein levels. The expression of HGF was downregulated by transforming growth factor beta. HGF stimulated chemotactic migration but not proliferation of hMSC. Therefore the HGF/c-met signaling system may have an important role in hMSC recruitment sites of tissue regeneration. The controlled regulation of HGF/c-met expression may be beneficial in tissue engineering and cell therapy employing hMSC.

Cell Differentiation↗
PubMed2002

Successful transduction of human multipotent, lymphoid (T, B, NK) and myeloid, and transplantable CD34+CD38low cord blood cells using a murine oncoretroviral vector.

Hematopoietic stem cells (HSC) are subject to great interest because of their medical importance and their biological properties. Therefore, the possibility of genetically modifying human HSC is a major concern in several inherited pathologies. In this study, we aimed to demonstrate that a murine oncoretroviral vector can transduce multipotential cord blood (CB) stem cells. Sorted CB CD34(+)CD38(low) cells were transduced with a Moloney-based MFG retroviral vector containing the coding sequence of the murine CD2 (mCD2). CD34(+)mCD2(+) cells were sorted by flow cytometry and cultured either in bulk or at one cell per well in culture conditions that allow differentiation along lymphoid (T, B, and NK) and myeloid (M) lineages. Phenotypic analysis of cells generated in culture showed that CD34(+)mCD2(+) cells could give rise to all lymphoid and myeloid progeny, indicating that the MFG/mCD2 vector had transduced progenitors of all tested lineages. Moreover, clonal cultures of 660 CD34(+)mCD2(+) cells showed that approximately 5% of these cells were able to generate both myeloid and lymphoid (B + NK) progenies; for 25% of them, this included the production of lymphoid T cells. We also demonstrate that transduced CD34(+)CD38(low) CB cells with lymphoid and myeloid potentials were capable of engraftment into the bone marrow (BM) of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice during several months. These results show that MFG retroviral vectors can transduce multipotent (T, B, NK, M) human hematopoietic progenitors with in vivo repopulating activity.

Animals↗
PubMed2006

Differentiation-related changes in mitochondrial properties as indicators of stem cell competence.

Several methods may be used to assess stem cell competence, including the expression of cell surface markers and telomerase activity. We hypothesized that mitochondrial characteristics might be an additional and reliable way to verify stem cell competence. In a multipotent, adult monkey stromal stem cell line, previously shown to differentiate into adipocytes, chondrocytes, and osteocytes, we found that several mitochondrial properties change with increasing passage number in culture. Cells from the earliest passage (P11) versus those from a later passage (P17) are characterized by: (a) a much higher percentage of cells (85% vs. 18%) with a perinuclear arrangement of mitochondria; (b) a much lower percentage of cells (1% vs. 57%) with an aggregated mitochondrial arrangement, in which mitochondria appear to coalesce into large clumps; (c) a much lower percentage of cells with lipid droplets (1% vs. 36%), suggesting less differentiation into adipocytes; (d) a 5.6-fold lower ATP content per cell (0.45 vs. 2.51 pmoles ATP/cell; and (e) a 10-fold higher rate of oxygen consumption (37.8 vs. 3.8 nmoles O2/min/10(3) cells), indicating a higher metabolic activity. Collectively, these data indicate that the perinuclear arrangement of mitochondria, accompanied by a low ATP/cell content and a high rate of oxygen consumption, may be valid indicators of stem cell differentiation competence, while departures from this profile indicate that cells are differentiating or perhaps becoming senescent. These results represent the first characterization of mitochondrial properties reported for a primate stem cell line.

Adenosine Triphosphate↗
PubMed2005

Human stem cells isolated from adult skeletal muscle differentiate into neural phenotypes.

Multipotent neural stem cells have been isolated from the adult [Kirschenbaum B, Nedergaard M, Preuss A, Barami K, Fraser RA, Goldman SA. In vitro neuronal production and differentiation by precursor cells derived from the adult human forebrain. Cereb Cortex 1994;4(6):576-89; Laywell ED, Kukekov VG, Steindler DA. Multipotent neurospheres can be derived from forebrain subependymal zone and spinal cord of adult mice after protracted postmortem intervals. Exp Neurol 1999;156:430-3; Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003;422:688-94] and embryonic [Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke E, et al. Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 1999;156:71-83] central nervous system (CNS). In addition, neural cells can be obtained from sources other than the CNS by differentiating stem cells from a non-neural source down a neural lineage. This has previously been performed with pluripotent embryonic stem cells and adult stem cells derived from rat bone marrow [Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364-70; Woodbury D, Reynolds K, Black IB. Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis. J Neurosci 2002;69(6):908-17] and skeletal muscle [Romero-Ramos M, Vourc'h P, Young HE, Lucas PA, Wu Y, Chivatakarn O, et al. Neuronal differentiation of stem cells isolated from adult muscle. J Neurosci Res 2002;69:894-907]. Previously, we have isolated adult stem cells from human skeletal muscle with the potential to differentiate into mesoderm, ectoderm, and endoderm. The following in vitro experiments were designed to determine whether human adult stem cells behaved similarly to rat adult stem cells when both were isolated from skeletal muscle by the same procedure [Romero-Ramos M, Vourc'h P, Young HE, Lucas PA, Wu Y, Chivatakarn O, et al. Neuronal differentiation of stem cells isolated from adult muscle. J Neurosci Res 2002;69:894-907] and subjected to the same protocols to induce neurogenesis. The neural phenotypes that were created through the neurococktail or neurosphere protocol were analyzed for neural characteristics through morphology and immunohistochemistry antibody labeling for proteins to neurons (RT-97, beta-tubulin III, NF-160, NF-200, and synapsin), oligodendrocytes (CNPase and RIP), and astrocytes (GFAP). A calcium uptake assay also showed response to the neuronal excitotoxic agent glutamic acid. In conclusion, the neural differentiated stem cells derived from adult skeletal muscle may be a less invasive alternative for the treatment of CNS disorders over CNS derived neural stem cells.

Adult↗
PubMed2006

Isolation and characterization of kidney-derived stem cells.

Acute kidney injury is followed by regeneration of damaged renal tubular epithelial cells. The purpose of this study was to test the hypothesis that renal stem cells exist in the adult kidney and participate in the repair process. A unique population of cells that behave in a manner that is consistent with a renal stem cell were isolated from rat kidneys and were termed multipotent renal progenitor cells (MRPC). Features of these cells include spindle-shaped morphology; self-renewal for >200 population doublings without evidence for senescence; normal karyotype and DNA analysis; and expression of vimentin, CD90 (thy1.1), Pax-2, and Oct4 but not cytokeratin, MHC class I or II, or other markers of more differentiated cells. MRPC exhibit plasticity that is demonstrated by the ability of the cells to be induced to express endothelial, hepatocyte, and neural markers by reverse transcriptase-PCR and immunohistochemistry. The cells can differentiate into renal tubules when injected under the capsule of an uninjured kidney or intra-arterially after renal ischemia-reperfusion injury. Oct4 expression was seen in some tubular cells in the adult kidney, suggesting these cells may be candidate renal stem cells. It is proposed that MRPC participate in the regenerative response of the kidney to acute injury.

Animals↗
PubMedDate not supplied

Adult stem cells--perspectives in treatment of metabolic diseases.

There is currently great excitement and expectation concerning the differential potential of adult stem cells or adult cells with capacity of differentiation. As the body of work concerning transdifferentiation of somatic stem cells and bone marrow derived stem cells grows, the number of critics increases steadily questioning the reliability of reported findings. So scientists are now challenged more and more to prove that resulting differentiated somatic cells originated from somatic adult stem cell through a transdifferentiation process. Phenomenons such as fusion of cells have to be ruled out and the origin of the differentiated cell has to be determined by specific techniques i.e. in situ hybridisation. Cellular mimicry through uptake of specific factors out of the medium is questioned to be the reason for cells staining positive for Insulin. Some multipotent adult stem cells can cross lineage boundaries and differentiate into somatic cells of other lineages after being relocated. Bone marrow cells have been described to have the greatest plasticity among adult stem cells regenerating damaged liver or myocardium. It has been proposed that the differentiation of bone marrow derived adult stem cells occurs naturally even in healthy organs as a physiologic process of tissue-regeneration. Others believe that organ damage is essential to induce transdifferentiation by release of organ specific microenvironmental factors. We here try to constitute necessary data which should be demonstrated to give substantial evidence for transdifferentiation of newly characterized cells including exclusion of fusion, phagocytosis or DNA uptake, description of the outset cell, differentiation into all three germ layers and functional parameters.

Adult↗
PubMed2006

Characterization of epidermal neural crest stem cell (EPI-NCSC) grafts in the lesioned spinal cord.

We have characterized in the contusion-lesioned murine spinal cord the behavior of acutely implanted epidermal neural crest stem cells (EPI-NCSC, formerly eNCSC). EPI-NCSC, a novel type of multipotent adult stem cell, are remnants of the embryonic neural crest. They reside in the bulge of hair follicles and have the ability to differentiate into all major neural crest derivatives (Sieber-Blum, M., Grim, M., Hu, Y.F., Szeder, V., 2004. Pluripotent neural crest stem cells in the adult hair follicle. Dev. Dyn. 231, 258-269). Grafted EPI-NCSC survived, integrated, and intermingled with host neurites in the lesioned spinal cord. EPI-NCSC were non-migratory. They did not proliferate and did not form tumors. Significant subsets expressed neuron-specific beta-III tubulin, the GABAergic marker glutamate decarboxylase 67 (GAD67), the oligodendrocyte marker, RIP, or myelin basic protein (MBP). Close physical association of non-neuronal EPI-NCSC with host neurites was observed. Glial fibrillary acidic protein (GFAP) immunofluorescence was not detected. Collectively, our data indicate that intraspinal EPI-NCSC demonstrate several desirable characteristics that may include local neural replacement and re-myelination.

Animals↗
PubMed2005

Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons.

We have recently shown that the expression of nestin, the neural stem cell marker protein, is expressed in bulge-area stem cells of the hair follicle. We used transgenic mice with GFP expression driven by the nestin regulatory element [nestin-driven GFP (ND-GFP)]. The ND-GFP stem cells give rise to the outer-root sheath of the hair follicle as well as an ND-GFP interfollicular vascular network. In this study, we demonstrate that ND-GFP stem cells isolated from the hair-follicle bulge area that are negative for the keratinocyte marker keratin 15 can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. These pluripotent ND-GFP stem cells are positive for the stem cell marker CD34, as well as keratin 15-negative, suggesting their relatively undifferentiated state. The apparent primitive state of the ND-GFP stem cells is compatible with their pluripotency. Furthermore, we show that cells derived from ND-GFP stem cells can differentiate into neurons after transplantation to the subcutis of nude mice. These results suggest that hair-follicle bulge-area ND-GFP stem cells may provide an accessible, autologous source of undifferentiated multipotent stem cells for therapeutic application.

Animals↗
PubMed2006

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction.

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.

Animals↗