Showing papers in "Stem Cells in 2004"
TL;DR: The differentiation of expanded adult human MSCs into cells with phenotypic and functional features of endothelial cells are shown to provide new options for engineering of artificial tissues based on autologous M SCs and vascularized engineered tissues.
Abstract: Human bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes, chondrocytes, and adipocytes in vivo and in vitro The aim of this study was to investigate the in vitro differentiation of MSCs into cells of the endothelial lineage MSCs were generated out of mononuclear bone marrow cells from healthy donors separated by density gradient centrifugation Cells were characterized by flow cytometry using a panel of monoclonal antibodies and were tested for their potential to differentiate along different mesenchymal lineages Isolated MSCs were positive for the markers CD105, CD73, CD166, CD90, and CD44 and negative for typical hematopoietic and endothelial markers They were able to differentiate into adipocytes and osteocytes after cultivation in respective media Differentiation into endothelial-like cells was induced by cultivation of confluent cells in the presence of 2% fetal calf serum and 50 ng/ml vascular endothelial growth factor Laser scanning cytometry analysis of the confluent cells in situ showed a strong increase of expression of endothelial-specific markers like KDR and FLT-1, and immunofluorescence analysis showed typical expression of the von Willebrand factor The functional behavior of the differentiated cells was tested with an in vitro angiogenesis test kit where cells formed characteristic capillary-like structures We could show the differentiation of expanded adult human MSCs into cells with phenotypic and functional features of endothelial cells These predifferentiated cells provide new options for engineering of artificial tissues based on autologous MSCs and vascularized engineered tissues
1,290 citations
TL;DR: It is shown that mesenchymal cells isolated from the umbilical cord have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages.
Abstract: The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells. Using flow cytometric analysis, we found that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45). Interestingly, these cells also express significant amounts of mesenchymal stem cell markers (SH2, SH3). We therefore investigated the potential of these cells to differentiate into cardiomyocytes by treating them with 5-azacytidine or by culturing them in cardiomyocyte-conditioned medium and found that both sets of conditions resulted in the expression of cardiomyocyte markers, namely N-cadherin and cardiac troponin I. We also showed that these cells have multilineage potential and that, under suitable culture conditions, are able to differentiate into cells of the adipogenic and osteogenic lineages. These findings may have a significant impact on studies of early human cardiac differentiation, functional genomics, pharmacological testing, cell therapy, and tissue engineering by helping to eliminate worrying ethical and technical issues.
1,283 citations
TL;DR: It is reported that second‐trimester amniotic fluid (AF) is an abundant source of fetal mesenchymal stem cells (MSCs), and different parts of the human placenta were studied for the presence of either fetal or maternal MSCs.
Abstract: Recently we reported that second-trimester amniotic fluid (AF) is an abundant source of fetal mesenchymal stem cells (MSCs). In this study, we analyze the origin of these MSCs and the presence of MSCs in human-term AF. In addition, different parts of the human placenta were studied for the presence of either fetal or maternal MSCs. We compared the phenotype and growth characteristics of MSCs derived from AF and placenta. Cells from human second-trimester (mean gestational age, 19(+2) [standard deviation, +/- 1(+3)] weeks, n = 10) and term third-trimester (mean gestational age, 38(+4) [standard deviation, +/- 1] weeks, n = 10) AF, amnion, decidua basalis, and decidua parietalis were cultured in M199 medium supplemented with 10% fetal calf serum and endothelial cell growth factor. Cultured cells were immunophenotypically characterized, the adipogenic and osteogenic differentiation capacity was tested, and the growth kinetics were analyzed. The origin of fetal and maternal cells was determined by molecular human leukocyte antigen typing. We successfully isolated MSCs from second-trimester AF, amnion, and decidua basalis as well as term amnion, decidua parietalis, and decidua basalis. In contrast, MSCs were cultured from only 2 out of 10 term AF samples. The phenotype of MSCs cultured from different fetal and maternal parts of the placenta was comparable. Maternal MSCs from second-trimester and term decidua basalis and parietalis showed a significantly higher expansion capacity than that of MSCs from adult bone marrow (p < .05). Our results indicate that both fetal and maternal MSCs can be isolated from the human placenta. Amnion is a novel source of fetal MSCs, likely contributing to the presence of MSCs in AF. Decidua basalis and decidua parietalis are sources for maternal MSCs. The expansion potency from both fetal and maternal placenta-derived MSCs was higher compared with adult bone marrow-derived MSCs.
1,196 citations
TL;DR: This work investigated whether cells with MSC characteristics and multi‐lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria.
Abstract: Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However, attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions, in up to 63% of 59 low-volume UCB units, cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition, osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However, in contrast to MSCs, MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours, a net volume of more than 33 ml, and an MNC count of more than 1 x 10(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations, we regard UCB as an additional stem cell source for experimental and potentially clinical purposes.
936 citations
TL;DR: The effect of in vitro expansion on the replicative capacity of MSCs is determined by correlating their rate of telomere loss during in vitro Expansion with their behavior in vivo, and it is reported that even protocols that involve minimal expansion induce a rapid aging of M SCs, with losses equivalent to about half their total replicative lifespan.
Abstract: Human marrow stromal cells (MSCs) can be isolated from bone marrow and differentiate into multiple tissues in vitro and in vivo. These properties make them promising tools in cell and gene therapy. The lack of a specific MSC marker and the low frequency of MSCs in bone marrow necessitate their isolation by in vitro expansion prior to clinical use. This may severely reduce MSC proliferative capacity to the point that the residual proliferative potential is insufficient to maintain long-term tissue regeneration upon reinfusion. In this study we determined the effect of in vitro expansion on the replicative capacity of MSCs by correlating their rate of telomere loss during in vitro expansion with their behavior in vivo. We report that even protocols that involve minimal expansion induce a rapid aging of MSCs, with losses equivalent to about half their total replicative lifespan.
728 citations
TL;DR: It is suggested that placenta‐derived cells have multilineage differentiation potential similar to MSCs in terms of morphology, cell‐surface antigen expression, and gene expression patterns.
Abstract: Mesenchymal stem/progenitor cells (MSCs) are widely distributed in a variety of tissues in the adult human body (e.g., bone marrow [BM], kidney, lung, and liver). These cells are also present in the fetal environment (e.g., blood, liver, BM, and kidney). However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and established two clones by limiting dilution. We examined these cells for morphology, surface markers, gene expression patterns, and differentiation potential and found that they expressed several stem cell markers, hematopoietic/ endothelial cell-related genes, and organ-specific genes, as determined by reverse transcription-polymerase chain reaction and fluorescence-activated cell sorter analysis. They also showed osteogenic and adipogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology, cell-surface antigen expression, and gene expression patterns. The placenta may prove to be a useful source of MSCs.
642 citations
TL;DR: It is shown that the LIFRβ and the signaling subunit gp130 are expressed in hESCs and that human LIF can induce STAT3 phosphorylation and nuclear translocation in h ESCs, Nevertheless, despite the functional activation of the Lif‐STAT3 signaling pathway, human Lif is unable to maintain the pluripotent state of hEScs.
Abstract: Murine embryonic stem (mES) cells remain undifferentiated in the presence of leukemia inhibitory factor (LIF), and activation of signal transducer and activator of transcription 3 (STAT3) via LIF receptor (LIFR) signaling appears sufficient for maintenance of mES cell pluripotency. Anecdotal and contradictory accounts exist for the action of LIF in the culture of human embryonic stem cells, and the nature of LIF signaling and whether the LIF-STAT3 pathway is conserved in human embryonic stem cells (hESCs) has not been systematically explored. In this study, we show that the LIFRbeta and the signaling subunit gp130 are expressed in hESCs and that human LIF can induce STAT3 phosphorylation and nuclear translocation in hESCs. Nevertheless, despite the functional activation of the LIF-STAT3 signaling pathway, human LIF is unable to maintain the pluripotent state of hESCs. Feeder-free culture conditions that maintain hESCs in an undifferentiated state do not show activation of STAT3, suggesting that distinct signaling mechanisms govern the self-renewal of hESCs.
541 citations
TL;DR: It is found that normal skin is a target organ for bone marrow–derived cells from both the hematopoietic and the mesenchymal stem cell pool, and wound healing involves local cutaneous cells for reconstituting the epidermis but distant bone marrow-derived cells and the adjacent uninjured dermal mesenchymeal cells for reconstructing the dermal fibroblast population.
Abstract: The bone marrow provides inflammatory cells and endothelial progenitor cells to healing cutaneous wounds. To further explore the bone marrow contribution to skin and healing wounds, we used a chimeric mouse model in which the bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice is transplanted into normal C57BL mice. We found that normal skin is a target organ for bone marrow–derived cells from both the hematopoietic and the mesenchymal stem cell pool. We present evidence that the bone marrow contribution to normal skin and the healing cutaneous wound is substantially greater than the previously recognized CD45 + sub-population, where 15%– 20% of the spindle-shaped dermal fibroblasts were bone marrow–derived (EGFP + ). Furthermore, the bone marrow–derived cells were able to contract a collagen matrix and transcribe both collagen types I and III, whereas the skin-resident cells transcribed only collagen type I. Whereas endothelial progenitor cells were found early during the wound repair process, bone marrow–derived endothelial cells were not seen after epithelialization was complete. Our data show that wound healing involves local cutaneous cells for reconstituting the epidermis but distant bone marrow–derived cells and the adjacent uninjured dermal mesenchymal cells for reconstituting the dermal fibroblast population.
484 citations
TL;DR: The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of β cells for transplantation for type I diabetes mellitus.
Abstract: Type I diabetes mellitus is caused by an autoimmune destruction of the insulin-producing beta cells. The major obstacle in using transplantation for curing the disease is the limited source of insulin-producing cells. The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of beta cells for transplantation. We present here a method for forming immature islet-like clusters of insulin-producing cells derived from hES cells. The protocol consisted of several steps. Embryoid bodies were first cultured and plated in insulin-transferrin-selenium-fibronectin medium, followed by medium supplemented with N2, B27, and basic fibroblast growth factor (bFGF). Next, the glucose concentration in the medium was lowered, bFGF was withdrawn, and nicotinamide was added. Dissociating the cells and growing them in suspension resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability than cells grown as monolayers. Reverse transcription-polymerase chain reaction detected an enhanced expression of pancreatic genes in the differentiated cells. Immunofluorescence and in situ hybridization analyses revealed a high percentage of insulin-expressing cells in the clusters. In addition to insulin, most cells also coexpressed glucagon or somatostatin, indicating a similarity to immature pancreatic cells. Further improvement of this insulin-producing cell protocol may lead to the formation of an unlimited source of cells suitable for transplantation.
444 citations
TL;DR: The overlapping similarities and differences in gene expression profiles of human and mouse ES cells provide a foundation for a detailed and concerted dissection of the molecular and cellular mechanisms governing their pluripotency, directed differentiation into specific cell types, and extended ability for self‐renewal.
Abstract: Human embryonic stem (ES) cell lines that have the ability to self-renew and differentiate into specific cell types have been established. The molecular mechanisms for self-renewal and differentiation, however, are poorly understood. We determined the transcriptome profiles for two proprietary human ES cell lines (HES3 and HES4, ES Cell International), and compared them with murine ES cells and other human tissues. Human and mouse ES cells appear to share a number of expressed gene products although there are numerous notable differences, including an inactive leukemia inhibitory factor pathway and the high preponderance of several important genes like POU5F1 and SOX2 in human ES cells. We have established a list of genes comprised of known ES-specific genes and new candidates that can serve as markers for human ES cells and may also contribute to the "stemness" phenotype. Of particular interest was the downregulation of DNMT3B and LIN28 mRNAs during ES cell differentiation. The overlapping similarities and differences in gene expression profiles of human and mouse ES cells provide a foundation for a detailed and concerted dissection of the molecular and cellular mechanisms governing their pluripotency, directed differentiation into specific cell types, and extended ability for self-renewal.
443 citations
TL;DR: In this paper, the role of some chemokines and their receptors in the trafficking of rat mesenchymal stem cells (rMSCs) in a rat model of left hypoglossal nerve injury was examined.
Abstract: Mesenchymal stem cells (MSCs), cultured ex vivo, recently were shown to be able to migrate into sites of brain injuries when transplanted systemically or locally, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. In this study, we examined the role of some chemokines and their receptors in the trafficking of rat MSCs (rMSCs) in a rat model of left hypoglossal nerve injury. rMSCs transplanted into the lateral ventricles of the rat brain migrated to the avulsed hypoglossal nucleus, where the expression of chemokines, stromal-cell-derived factor 1 (SDF-1), and fractalkine was observed to be increased. This increase temporally paralleled the migration of rMSCs into the avulsed nucleus at 1 and 2 weeks after operation. It has been found that rMSCs express CXCR4 and CX3CR1, the respective receptors for SDF-1 and fractalkine, and other chemokine receptors, CCR2 and CCR5. Furthermore, in vitro analysis revealed that recombinant human SDF-1 alpha (rhSDF-1alpha) and recombinant rat fractalkine (rrfractalkine) induced the migration of rMSCs in a G-protein-dependent manner. Intracerebral injection of rhSDF-1alpha has also been shown to stimulate the homing of transplanted rMSCs to the site of injection in the brain. These data suggest that the interactions of fractalkine-CX3CR1 and SDF-1-CXCR4 could partially mediate the trafficking of transplanted rMSCs. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted rMSCs and also significantly expands the potential role of MSCs in cell therapy for brain injuries and diseases.
TL;DR: This work suggests that the basal epithelial cells of the limbus are p63, ABCG2 and integrin α9 positive, and nestin, E‐cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin β1, EGFR, K19, and enolase‐α.
Abstract: This study evaluated proposed molecular markers related to stem cell (SC) properties with the intention of characterizing a putative SC phenotype in human limbal epithelia. Human corneal and limbal tissues were cut in the vertical and horizontal meridians for histology, transmission electron microscopy (TEM), and immunostaining. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization were used to evaluate gene expression. TEM showed that the limbal basal cells were small primitive cells. Immunostaining disclosed that p63, ABCG2 and integrin α9 were primarily expressed by the basal epithelial cells of limbus. Antibodies against integrin β1, epidermal growth factor receptor (EGFR), K19, enolase-α, and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin α6, nestin, E-cadherin and connexin 43 did not stain the limbal basal cells, but the suprabasal epithelia of the cornea and limbus showed strong immunoreactivity. K3 and involucrin stained only corneal and limbal superficial cells. RT-PCR showed higher levels of p63, ABCG2 and integrin α9 mRNA, but lower levels of K3, K12 and connexin 43 expressed in the limbal epithelia than the corneal epithelia. In situ hybridization showed that p63 transcripts were located in basal layer of the limbal epithelium. This work suggests that the basal epithelial cells of the limbus are p63, ABCG2 and integrin α9 positive, and nestin, E-cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin β1, EGFR, K19, and enolase-α. This putative SC phenotype may facilitate the identification and isolation of limbal epithelial SCs.
TL;DR: The general requirement for Oct‐4 in maintaining pluripotency in ES cells is established, and the upregulation of endoderm‐associated markers in both mouse and human ES cells points to overlap between development of trophoblast and endODerm differentiation.
Abstract: The transcription factor Oct-4 is a marker of pluripotency in mouse and human embryonic stem (ES) cells. Previous studies using a tetracycline-regulated Oct-4 transgene in the ZHBTc4 cell line demonstrated that downregulation of Oct-4 expression induced dedifferentiation into trophoblast, a lineage mouse ES cells do not normally generate. We found that transfection of Oct-4-specific short interfering RNA significantly reduced expression and functional activity of Oct-4 in mouse and human ES cells, enabling its role to be compared in both cell types. In mouse ES cells, Oct-4 knockdown produced a pattern of morphological differentiation and increase in expression of the trophoblast-associated transcription factor Cdx2, similar to that triggered by suppressing the Oct-4 transgene in the ZHBTc4 cell line. In addition, downregulation of Oct-4 was accompanied by increased expression of the endoderm-associated genes Gata6 and α-fetoprotein, and a gene trap associated with primitive liver/yolk sac differentiation. In human ES cells, Oct-4 knockdown also induced morphological differentiation coincident with the upregulation of Gata6. The induction of Cdx2 and other trophoblast-associated genes, however, was dependent on the culture conditions. These results establish the general requirement for Oct-4 in maintaining pluripotency in ES cells. Moreover, the upregulation of endoderm-associated markers in both mouse and human ES cells points to overlap between development of trophoblast and endoderm differentiation.
TL;DR: It is suggested that cyclic compressive loading can promote the chondrogenesis of rabbit BM‐MSCs by inducing the synthesis of TGF‐β1, which can stimulate the BM‐ MSCs to differentiate into chondrocytes.
Abstract: The objective of this study was to examine the effects of cyclic compressive loading on chondrogenic differentiation of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures. Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand white rabbits. After the chondrogenic potential of BM-MSCs was verified by pellet cultures, cell-agarose constructs were made by suspending BM-MSCs in 2% agarose (10(7) cells/ml) for a cyclic, unconfined compression test performed in a custom-made bioreactor. Specimens were divided into four groups: control; transforming growth factor (TGF-beta) (with TGF-beta1 treatment); loading (with stimulation of cyclic, unconfined compressive loading); and TGF-beta loading (with TGF-beta1 treatment and loading stimulation) groups. In the loading experiment, specimens were subjected to sinusoidal loading with a 10% strain magnitude at a frequency of 1 Hz for 4 hours a day. Experiments were conducted for 3, 7, and 14 consecutive days. While the experimental groups (TGF-beta, loading, and TGF-beta loading) exhibited significantly higher levels of expressions of chondrogenic markers (collagen II and aggrecan) at three time periods, there were no differences among the experimental groups after an extra 5-day culture. This suggests that compressive loading alone induces chondrogenic differentiation of rabbit BM-MSCs as effectively as TGF-beta or TGF-beta plus loading treatment. Moreover, both the compressive loading and the TGF-beta1 treatment were found to promote the TGF-beta1 gene expression of rabbit BM-MSCs. These findings suggest that cyclic compressive loading can promote the chondrogenesis of rabbit BM-MSCs by inducing the synthesis of TGF-beta1, which can stimulate the BM-MSCs to differentiate into chondrocytes.
TL;DR: The literature that lends credibility to the theory that highly plastic BMCs have a role in maintenance and repair of nonhematopoietic tissue is reviewed and the possible mechanisms by which this may occur are discussed.
Abstract: Stem cell plasticity refers to the ability of adult stem cells to acquire mature phenotypes that are different from their tissue of origin. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into bone, cartilage, and fat. In this article, we review the literature that lends credibility to the theory that highly plastic BMCs have a role in maintenance and repair of nonhematopoietic tissue. We discuss the possible mechanisms by which this may occur. Also reviewed is the possibility that adult BMCs can change their gene expression profile after fusion with a mature cell, which has brought into question whether this stem cell plasticity is real.
TL;DR: It is demonstrated that the agglomeration of two EBs is initiated by E‐cadherin‐mediated cell attachment and followed by active cell migration, which enables the development of a technology capable of controlling cell‐cell interactions in scalable culture by the mass encapsulation of ES cells in size‐specified agarose capsules.
Abstract: Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subsequent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high-cell-density static cultures. We demonstrate that the agglomeration of two EBs is initiated by E-cadherin-mediated cell attachment and followed by active cell migration. We report the development of a technology capable of controlling cell-cell interactions in scalable culture by the mass encapsulation of ES cells in size-specified agarose capsules. When placed in stirred-suspension bioreactors, encapsulated ES cells can be used to produce scalable quantities of hematopoietic progenitor cells in a controlled environment.
TL;DR: It is reported that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells, and a high percentage of the cells in most of the colonies expressed TH.
Abstract: In this manuscript we report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)–positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum.
TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine-β-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K+.
When transplanted into 6-hydroxydopamine–treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons.
TL;DR: It is reported that STAT3 activation is not sufficient to block hES cell differentiation when the cells are grown on mouse feeder cells or when they are treated with conditioned media from feedercells, and the existence of an as‐yet‐unidentified factor in the conditioned media of mouseFeeder layer cells that acts to maintain h ES cell renewal in a STAT3‐independent manner is demonstrated.
Abstract: The preservation of "stemness" in mouse embryonic stem (mES) cells is maintained through a signal transduction pathway that requires the gp130 receptor, the interleukin-6 (IL-6) family of cytokines, and the Janus Kinase-signal transducer and activator (JAK/STAT) pathway. The factors and signaling pathways that regulate "stemness" in human embryonic stem (hES) cells remain to be elucidated. Here we report that STAT3 activation is not sufficient to block hES cell differentiation when the cells are grown on mouse feeder cells or when they are treated with conditioned media from feeder cells. Human ES cells differentiate in the presence of members of the IL-6 family of cytokines including leukemia inhibitory factor (LIF) and IL-6 or in the presence of the designer cytokine hyper-IL-6, which is a complex of soluble interleukin-6 receptor (IL-6R) and IL-6 with greatly enhanced bioactivity. Human ES cells express LIF, IL-6, and gp130 receptors, as well as the downstream signaling molecules. Stimulation of human and mouse ES cells with gp130 cytokines resulted in a robust phosphorylation of downstream ERK1, ERK2, and Akt kinases, as well as the STAT3 transcription factor. Loss of the pluripotency markers Nanog, Oct-4, and TRA-1-60 was observed in hES cells during gp130-dependent signaling, indicating that signaling through this pathway is insufficient to prevent the onset of differentiation. These data underscore a fundamental difference in requirements of murine versus hES cells. Furthermore, the data demonstrate the existence of an as-yet-unidentified factor in the conditioned media of mouse feeder layer cells that acts to maintain hES cell renewal in a STAT3-independent manner.
TL;DR: The data indicate that the host‐lesioned striatum could not direct the transplanted neural progenitors to acquire a dopaminergic fate, and induction of their differentiation toward a midbrain fate prior to transplantation is probably required for complete correction of behavioral deficit.
Abstract: Human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for transplantation. In Parkinson's disease, hESC-derived dopaminergic (DA) neurons may replace the degenerated neurons in the brain. Here, we generated highly enriched cultures of neural progenitors from hESCs and grafted the progenitors into the striatum of Parkinsonian rats. The grafts survived for at least 12 weeks, the transplanted cells stopped proliferating, and teratomas were not observed. The grafted cells differentiated in vivo into DA neurons, though at a low prevalence similar to that observed following spontaneous differentiation in vitro. Transplanted rats exhibited a significant partial correction of D-amphetamine and apomorphine-induced rotational behavior, along with a significant improvement in stepping and placing non-pharmacological behavioral tests. While transplantation of uncommitted hESC-derived neural progenitors induced partial behavioral recovery, our data indicate that the host-lesioned striatum could not direct the transplanted neural progenitors to acquire a dopaminergic fate. Hence, induction of their differentiation toward a midbrain fate prior to transplantation is probably required for complete correction of behavioral deficit. Our observations encourage further developments for the potential use of hESCs in the treatment of Parkinson's disease.
TL;DR: It is demonstrated that hESCs possess unique immune‐privileged characteristics and provides an unprecedented opportunity to further investigate the mechanisms of immune response to transplantation of hESC banks that may avoid immune‐mediated rejection.
Abstract: Human embryonic stem cells (hESCs) are envisioned to be a major source for cell-based therapies. Efforts to overcome rejection of hESCs include nuclear transfer and collection of hESC banks representing the broadest diversity of major histocompatability complex (MHC) polymorphorisms. Surprisingly, immune responses to hESCs have yet to be experimentally evaluated. Here, injection of hESCs into immune-competent mice was unable to induce an immune response. Undifferentiated and differentiated hESCs failed to stimulate proliferation of alloreactive primary human T cells and inhibited third-party allogeneic dendritic cell-mediated T-cell proliferation via cellular mechanisms independent of secreted factors. Upon secondary rechallenge, T cells cocultured with hESCs were still responsive to allogeneic stimulators but failed to proliferate upon re-exposure to hESCs. Our study demonstrates that hESCs possess unique immune-privileged characteristics and provides an unprecedented opportunity to further investigate the mechanisms of immune response to transplantation of hESCs that may avoid immune-mediated rejection.
TL;DR: MSCs isolated from UC veins are functionally similar to BM‐MSCs, but differentially expressed genes may reflect differences related to their sites of origin: BM‐ MSCs would be more committed to osteogenesis, whereas UC‐MSC would beMore committed to angiogenesis.
Abstract: Mesenchymal stem cells (MSCs) give origin to the marrow stromal environment that supports hematopoiesis. These cells present a wide range of differentiation potentials and a complex relationship with hematopoietic stem cells (HSCs) and endothelial cells. In addition to bone marrow (BM), MSCs can be obtained from other sites in the adult or the fetus. We isolate MSCs from the umbilical cord (UC) veins that are morphologically and immunophenotpically similar to MSCs obtained from the BM. In culture, these cells are capable of differentiating in vitro into adipocytes, osteoblasts, and condrocytes. The gene expression profiles of BM-MSCs and of UC-MSCs were compared by serial analysis of gene expression, then validated by reverse transcription polymerase chain reaction of selected genes. The two lineages shared almost all of the first thousand most expressed transcripts, including vimentin, galectin 1, osteonectin, collagens, transgelins, annexin A2, and MMP2. Nevertheless, a set of genes related to antimicrobial activity and to osteogenesis was more expressed in BM-MSCs, whereas higher expression in UC-MSCs was observed for genes that participate in pathways related to matrix remodeling via metalloproteinases and angiogenesis. Finally, cultured endothelial cells, CD34+ HSCs, MSCs, blood leukocytes, and bulk BM clustered together, separated from seven other normal nonhematopoietic tissues, on the basis of shared expressed genes. MSCs isolated from UC veins are functionally similar to BM-MSCs, but differentially expressed genes may reflect differences related to their sites of origin: BM-MSCs would be more committed to osteogenesis, whereas UC-MSCs would be more committed to angiogenesis.
TL;DR: A consistent expression profile of Wnt signaling molecules in MSCs is identified and evidence that an endogenous canonical Wnt pathway functions in these cells is provided.
Abstract: Through their broad differentiation potential, mesenchymal stem cells (MSCs) are candidates for a range of therapeutic applications, but the precise signaling pathways that determine their differentiated fate are not fully understood. Evidence is emerging that developmental signaling cues may be important in regulating stem cell self-renewal and differentiation programs. Here we have identified a consistent expression profile of Wnt signaling molecules in MSCs and provide evidence that an endogenous canonical Wnt pathway functions in these cells. Wnts bind to Frizzled (Fz) receptors and subsequent canonical signaling inhibits glycogen synthase kinase-3beta (GSK-3beta), causing beta-catenin translocation into the nucleus to induce target gene expression. In human MSCs isolated from bone marrow of different donors, we appear to have identified a common Wnt/Fz expression profile using reverse transcriptase polymerase chain reaction (RT-PCR). Associated Wnt signaling components, including low-density lipoprotein receptor-related protein-5 (LRP-5), kremen-1, dickkopf-1 (Dkk-1), secreted Frizzled-related peptide (sFRP)-2, sFRP3, sFRP4, Disheveled (Dvl), GSK-3beta, adenomatous polyposis coli (APC), beta-catenin,T-cell factor (TCF)-1, and TCF-4, were also identified. Nuclear beta-catenin was observed in 30%-40% of MSCs, indicative of endogenous Wnt signaling. Exposure to both Wnt3a and Li+ ions, which promotes canonical Wnt signaling by inhibiting GSK-3beta, reduced phosphorylation of beta-catenin in MSCs and increased beta-catenin nuclear translocation approximately threefold over that of the controls. Our findings indicate that autocrine Wnt signaling operates in primitive MSC populations and supports previous evidence that Wnt signaling regulates mesenchymal lineage specification. The identification of a putative common Wnt/Fz molecular signature in MSCs will contribute to our understanding of the molecular mechanisms that regulate self-renewal and lineage-specific differentiation.
TL;DR: The derivation and characterization of six hES cell lines were presented and one cell line was clonally derived, which could be propagated in an undifferentiated pluripotent state.
Abstract: The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.
TL;DR: It is reported that hepatocyte growth factor (HGF) and the cognate receptor HGFR/c‐met are expressed in hMSC, on both the RNA and the protein levels, and the HGF/ c‐met signaling system may have an important role in h MSC recruitment sites of tissue regeneration.
Abstract: 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.
TL;DR: The results demonstrate that UCB‐derived MSCs possess a potential of skeletal myogenic differentiation and also imply that these cells could be a suitable source for skeletal muscle repair and a useful tool of muscle‐related tissue engineering.
Abstract: Human umbilical cord blood (UCB) has been regarded as an alternative source for cell transplantation and cell therapy because of its hematopoietic and nonhematopoietic (mesenchymal) potential. Although there has been debate about whether mesenchymal stem cells (MSCs) are invariably present in UCB, several reports showed that MSC-like cells could be consistently derived from human UCB and, moreover, could differentiate into various cells of a mesodermal origin. However, it remains unclear whether these UCB-derived MSCs are also capable of differentiating into skeletal muscle cells. In this study, we isolated MSCs from human UCB and induced them to differentiate into skeletal muscle cells. During cell culture expansion, UCB-derived mononuclear cells gave rise to adherent layers of fibroblast-like cells expressing MSC-related antigens such as SH2, SH3, αsmooth muscle actin, CD13, CD29, and CD49e. More important, when these UCB-derived MSCs were incubated in promyogenic conditions for up to 6 weeks, they expressed myogenic markers in accordance with myogenic differentiation pattern. Both flow cytometric and reverse transcriptase‐polymerase reaction analyses showed that two early myogenic markers, MyoD and myogenin, were expressed after 3 days of incubation but not after 2 weeks. At week 6, more than half of UCBderived MSCs expressed myosin heavy chain, a late myogenic marker. Our results demonstrate that UCB-derived MSCs possess a potential of skeletal myogenic differentiation and also imply that these cells could be a suitable source for skeletal muscle repair and a useful tool of muscle-related tissue engineering. Stem Cells 2004; 22:617‐624
TL;DR: RNA interference is used to downregulate β2‐microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells.
Abstract: We have used RNA interference (RNAi) to downregulate β2-microglobulin and Oct4 in human embryonal carcinoma (hEC) cells and embryonic stem (hES) cells, demonstrating that RNAi is an effective tool for regulating specific gene activity in these human stem cells. The knockdown of Oct4 but not β2-microglobulin expression in both EC and ES cells resulted in their differentiation, as indicated by a marked change in morphology, growth rate, and surface antigen phenotype, with respect to SSEA1, SSEA3, and TRA-1-60 expression. Expression of hCG and Gcm1 was also induced following knockdown of Oct4 expression, in both 2102Ep hEC cells and in H7 and H14 hES cells, consistent with the conclusion that, as in the mouse, Oct4 is required to maintain the undifferentiated stem cell state, and that differentiation to trophectoderm occurs in its absence. NTERA2 hEC cells also differentiated, but not to trophectoderm, suggesting their equivalence to a later stage of embryogenesis than other hEC and hES cells.
TL;DR: Using microarrays and confirmation by reverse transcription–polymerase chain reaction, several candidate antioxidant and stress‐resistance genes that become downregulated during differentiation of ES cells into embryoid bodies are identified.
Abstract: A very small number of embryonic stem (ES) cells gives rise to all tissues of the embryo proper. This means that ES cells should be equipped with highly efficient mechanisms to defend themselves against various stresses and to prevent or repair DNA damage. One of these mechanisms is a high activity of a verapamil-sensitive multidrug efflux pump. Because reactive oxygen species are a major source of DNA damage, we further tested the idea that murine ES cells might differ from their more differentiated counterparts by high levels of antioxidant defense and good DNA strand break repair capacity. This was confirmed by comparing cellular peroxide levels, total antioxidant capacity, and activity of radiation-induced strand break repair between murine ES cells and embryoid bodies or embryonic fibroblasts. Using microarrays and confirmation by reverse transcription-polymerase chain reaction, we identified several candidate antioxidant and stress-resistance genes that become downregulated during differentiation of ES cells into embryoid bodies.
TL;DR: This work suggests that hESCs can differentiate in simple serum‐free suspension cultures to produce the large number of cells required for transplantation studies.
Abstract: The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.
TL;DR: This review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro and suggests that pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage‐related biomaterials and drugs could also utilize protocols developed for the chONDrogenic differentiation ofstem cells.
Abstract: A major area in regenerative medicine is the application of stem cells in cartilage tissue engineering and reconstructive surgery This requires well-defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage, followed by their selective purification and proliferation in vitro The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells Additionally, such protocols could provide useful in vitro models for studying chondrogenesis and cartilaginous tissue biology The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage-related biomaterials and drugs could also utilize protocols developed for the chondrogenic differentiation of stem cells Hence, this review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro
TL;DR: It is concluded that the Oct4 reprogramming capacity resides in the ESC karyoplast and that gene reactivation is independent of DNA replication and cell division.
Abstract: The restricted potential of a differentiated cell can be reverted back to a pluripotent state by cell fusion; totipotency can even be regained after somatic cell nuclear transfer. To identify factors involved in resetting the genetic program of a differentiated cell, we fused embryonic stem cells (ESCs) with neurosphere cells (NSCs). The fusion activated Oct4, a gene essential for pluripotency, in NSCs. To further identify whether cytoplasmic or nuclear factors are responsible for its reactivation, we fused either karyoplasts or cytoplasts of ESCs with NSCs. Our results show that ESC karyoplasts could induce Oct4 expression in the somatic genome, but cytoplasts lacked this ability. In addition, mitomycin C-treated ESCs, although incapable of DNA replication and cell division, could reprogram 5-azacytidine-treated NSCs. We therefore conclude that the Oct4 reprogramming capacity resides in the ESC karyoplast and that gene reactivation is independent of DNA replication and cell division.