Direct Stem Cell Differentiation Research Articles

Vertebrate Endoderm Development and Organ Formation

The endoderm germ layer contributes to the respiratory and gastrointestinal tracts and to all of their associated organs. Over the past decade, studies in vertebrate model organisms, including frog, fish, chick, and mouse, have greatly enhanced our understanding of the molecular basis of endoderm organ development. We review this progress with a focus on early stages of endoderm organogenesis including endoderm formation, gut tube morphogenesis and patterning, and organ specification. Lastly, we discuss how developmental mechanisms that regulate endoderm organogenesis are used to direct differentiation of embryonic stem cells into specific adult cell types, which function to alleviate disease symptoms in animal models.

Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells

Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells.

Transcription factor directed differentiation of embryonic stem cells in vitro

OBJECTIVE: We hypothesize that Pax6 and Six3 transcription factors (TF) that play roles critical in formation of the vertebrate ocular lens, may also facilitate directed differentiation of embryonic stem cells (ESC) to generate lens tissue in vitro. DESIGN: Mammalian lens placode development is regulated by key TFs, Pax6, a paired box gene in conjunction with the homeobox gene Six3. Loss-of-function mutations of PAX6 results in the ocular defects aniridia and anophthalmia in humans and the Small eye (Sey) phenotype in mice which includes diminished lens development and cataracts. We therefore chose to test whether Pax6 and Six3 can direct ESC differentiation to a lens fate in vitro. MATERIALS AND METHODS: G4 mouse ESCs and H1 NIH human ESCs were used in these studies. Plasmids or lentiviral vectors for Pax6 and Six3 were employed for transfection and infection of cultured mESC or hESC. Post transduction cultured cells were followed for 7 or 14 days and analyzed by immuncytochemistry, RT-PCR and protein analysis. RESULTS: We observed that transduction of mouse or human ESC with either Pax6 or Six3 genes drives differentiation of ESCs along the lens forming pathway. This was apparent from a near 40-fold increase in the number of γ-crystallin immunoreactive colonies, a lens terminal antigen, in Pax6 or Six3 transduced ESC colonies after 14 days. Furthermore, γ-crystallin expressing colonies formed distinct multicellular rosettes. Additional lens proteins such as Prox1 were also upregulated in the experimental cultures. Expression of other markers associated with lens development and differentiation such as Bmp7, Foxe3, Mab2111, Gja8, α-crystallin and β-crystallin was confirmed by RT-PCR and Western blot analysis. CONCLUSIONS: The lens endogenous TFs, Pax6 and Six3, can program mouse and human ESCs to similar fates in vitro. This suggests a potential route for using specific TFs to direct the efficient differentiation of ESCs to desired tissue fates in vitro.

Research Highlights

Research Highlights

14-P024 Physiologic and defined directed differentiation of human embryonic stem cells and human endodermal cultures to hepatocytes

14-P024 Physiologic and defined directed differentiation of human embryonic stem cells and human endodermal cultures to hepatocytes

Noggin Elicits Retinal Fate inXenopusAnimal Cap Embryonic Stem Cells

Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Here we exploited the differentiating potential of Xenopus animal cap embryonic stem (ACES) cells to investigate the factors necessary to drive multipotent stem cells toward retinal fates. ACES cells are multipotent, and can be diverged from their default ectodermal fate to give rise to cell types from all three germ layers. We found that a single secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES cells. Reverse-transcription polymerase chain reaction, immunohistochemistry, and in situ hybridization experiments showed that high doses of Noggin are able to support the expression of terminal differentiation markers of the neural retina in ACES cells in vitro. Following in vivo transplantation, ACES cells expressing high Noggin doses form eyes, both in the presumptive eye field region and in ectopic posterior locations. The eyes originating from the transplants in the eye field region are functionally equivalent to normal eyes, as seen by electrophysiology and c-fos expression in response to light. Our data show that in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without additional cues. This makes Xenopus ACES cells a suitable model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells.

Contribution of human hematopoietic stem cells to liver repair

Immune-deficient mouse models of liver damage allow examination of human stem cell migration to sites of damage and subsequent contribution to repair and survival. In our studies, in the absence of a selective advantage, transplanted human stem cells from adult sources did not robustly become hepatocytes, although some level of fusion or hepatic differentiation was documented. However, injected stem cells did home to the injured liver tissue and release paracrine factors that hastened endogenous repair and enhanced survival. There were significantly higher levels of survival in mice with a toxic liver insult that had been transplanted with human stem cells but not in those transplanted with committed progenitors. Transplantation of autologous adult stem cells without conditioning is a relatively safe therapy. Adult stem cells are known to secrete bioactive factors that suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate recruitment, retention, mitosis, and differentiation of tissue-residing stem cells. These paracrine effects are distinct from the direct differentiation of stem cells to repair tissue. In patients at high risk while waiting for a liver transplant, autologous stem cell therapy could be considered, as it could delay the decline in liver function.

Adult stem cells as a renewable source of insulin-producing cells.

Diabetes mellitus is a metabolic disorder resulting from an inadequate mass of insulin-producing pancreatic beta cells. The replacement or restoration of damaged beta cells would be considered the optimal therapeutic options. Islet transplantation seems to be a promising approach for replacement therapy; however, the main obstacle is the shortage of organ donors. As mature beta cells have been shown to be difficult to expand in vitro, regeneration of beta cells from embryonic or adult stem cells or pancreatic progenitor cells is an attractive method to restore the islet cell mass. So far, multiple studies using various strategies have shown direct differentiation of stem and progenitor cells toward insulin-producing cells. The important issue to be solved is how to differentiate these cells into mature functional insulin-producing cells. Further research is required to understand how endogenous beta cells differentiate and to develop methods to regenerate enough functional beta cells for clinically applicable therapies for diabetes.

Glandular tissue from human pancreas and salivary gland yields similar stem cell populations

Stem cells derived from pancreatic tissue are well characterized and exhibit a broad plasticity as they can differentiate beyond lineage boundaries into many cell types. The aim of this study was the comparative characterization of pancreatic stem cells with one other derivate of the embryonic foregut, namely salivary glands, for the existence of similar stem cell populations. The expression of stem cell markers as well as lineage-specific markers was detected by reverse transcription polymerase chain reaction, flow cytometry and immuncytochemical staining. The isolated cells from salivary glands and pancreas grew adherently in vitro and could be maintained for up to 55 and 46 population doublings, respectively. Cells from both tissues showed a comparable phenotype. They expressed different embryonic and adult stem cell markers and had the ability to differentiate spontaneously into cells representing the three embryonic germ layers. Additionally, the directed differentiation of glandular stem cells into the mesodermal lineage was achieved, yielding adipogenic, osteogenic and chondrogenic cells from salivary gland stem cells as well as osteogenic and chondrogenic cells from pancreatic stem cells. Here, we compared two stem cell populations from different glandular tissues which showed similar phenotypes and analogous properties. During embryonic development the two exocrine glands originate from the foregut, which might be the explanation for these intriguing resemblances.

Small Molecules Efficiently Direct Endodermal Differentiation of Mouse and Human Embryonic Stem Cells

An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver, and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved by Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos, and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives.

Pre-transplantation Specification of Stem Cells to Cardiac Lineage for Regeneration of Cardiac Tissue

Myocardial infarction (MI) is a lead cause of mortality in the Western world. Treatment of acute MI is focused on restoration of antegrade flow which inhibits further tissue loss, but does not restore function to damaged tissue. Chronic therapy for injured myocardial tissue involves medical therapy that attempts to minimize pathologic remodeling of the heart. End stage therapy for chronic heart failure (CHF) involves inotropic therapy to increase surviving cardiac myocyte function or mechanical augmentation of cardiac performance. Not until the point of heart transplantation, a limited resource at best, does therapy focus on the fundamental problem of needing to replace injured tissue with new contractile tissue. In this setting, the potential for stem cell therapy has garnered significant interest for its potential to regenerate or create new contractile cardiac tissue. While to date adult stem cell therapy in clinical trials has suggested potential benefit, there is waning belief that the approaches used to date lead to regeneration of cardiac tissue. As the literature has better defined the pathways involved in cardiac differentiation, preclinical studies have suggested that stem cell pretreatment to direct stem cell differentiation prior to stem cell transplantation may be a more efficacious strategy for inducing cardiac regeneration. Here we review the available literature on pre-transplantation conditioning of stem cells in an attempt to better understand stem cell behavior and their readiness in cell-based therapy for myocardial regeneration.

ECAM: Integrative Genomics and Fecundity

<i>eCAM</i>: Integrative Genomics and Fecundity

Human Cord Blood Progenitors with High Aldehyde Dehydrogenase Activity Improve Vascular Density in a Model of Acute Myocardial Infarction

Human stem cells from adult sources have been shown, in our laboratory and others, to promote tissue repair. Different populations of stem cells have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature, although the mechanisms by which they accomplish this are still not well understood. Stem cells are known, however, to secrete a variety of factors that have both paracrine and autocrine activities. One theory of tissue repair and regeneration by adult stem cells is that they home to hypoxic and/or inflamed areas, and release trophic factors that hasten endogenous repair. These secreted bioactive factors suppress the local immune system, enhance angiogenesis, inhibit fibrosis and apoptosis, and stimulate recruitment, retention, mitosis and differentiation of tissueresiding stem cells. These effects, which are referred to as trophic effects, are distinct from the direct differentiation of stem cells into the tissue to be regenerated. We tested human umbilical cord blood (UCB) derived CD34+, ALDHhiLin− and ALDHloLin− cells following transplantation to NOD/SCID or NOD/SCID/B2M null mice with experimentally induced acute myocardial infarction (AMI). We used combined nanoparticle labeling and whole organ fluorescent imaging to detect homing of the cells to multiple organs 48 hours post transplantation. Long term engraftment and the regenerative effect of cell treatment was assessed four weeks post transplant. There was superior homing of ALDHhiLin− cells to the site of injury, as compared to CD34+ or ALDHloLin− cells at 48 hours post transplantation. At four weeks post transplantation, ALDHhiLin− cells engrafted multiple organs, including the heart, liver and kidney, at higher frequencies than ALDHloLin− cells. We found no donor derived cardiomyocytes and only few endothelial cells of donor origin. However, there was a significant increase in the density of large caliber vessels in the central infarct zone of ALDHhiLin− cell-transplanted mice, as compared to PBS and ALDHloLin− cell treated groups. Tissue staining in the damaged regions revealed that the transplanted human cells had undergone very few cell divisions after homing to the area of hypoxia or inflammation. It is important to note that, with over 300 mice now analyzed in tissue repair experiments in our group, no adult human stem cell-derived tumors have ever been observed, even though the mouse strains used have no capacity to reject the human cells. The paucity of human cells remaining in the tissue after repair suggest that the tissue improvements that were observed were not the result of generation of transplanted cell-derived endothelial cells or cardiac tissue, but suggest that cytokines secreted from transplanted cells potentiated angiogenic activity and tissue repair by the endogenous murine cells. Our data indicate that adult human stem cells do not become a significant part of the damaged tissue, but rapidly home to and persist only temporarily at a site of hypoxia or inflammation to exert significant trophic effects on tissue repair, and to enhance recovery of the vasculature.

Recreating a Human Hematopoietic Stem Cell Niche in Vitro by Unique Stroma Cells from the First Trimester Human Placenta and Fetal Liver

Expansion of human hematopoietic stem cells (HSCs) ex vivo has been difficult due to limited understanding of their growth requirements and the molecular complexity of their natural microenvironments. To mimic the niches in which human HSCs normally develop and expand during ontogeny, we have derived two unique types of stromal niche cells from the first trimester human placenta and the fetal liver. These lines either support maintenance of multipotential progenitors in culture, or promote differentiation into macrophages. Impressively, the supportive lines facilitate over 50,000-fold expansion of the most immature human HSCs/progenitors (CD34+CD38-Thy1+) during 8-week culture supplemented with minimal cytokines FLT3L, SCF and TPO, whereas the cells cultured on non-supportive stroma or without stroma under the same conditions differentiated within 2 weeks. As the supportive stroma lines also facilitate differentiation of human hematopoietic progenitors into myeloid, erythroid and B-lymphoid lineages, we were able to show that the expanded progenitors preserved full multipotentiality during long-term culture ex vivo. Furthermore, our findings indicate that the supportive stroma lines also direct differentiation of human embryonic stem cells (hESC) into hematopoietic progenitor cells (CD45+CD34+) that generate multiple types of myeloerythroid colonies. These data imply that the unique supportive niche cells can both support hematopoietic specification and sustain a multilineage hematopoietic hierarchy in culture over several weeks. Strikingly, the supportive effect from the unique stromal cells was dominant over the differentiation effect from the non-supportive lines. Even supernatant from the supportive lines was able to partially protect the progenitors that were cultured on the non-supportive lines, whereas mixing of the two types of stroma resulted in sustained preservation of the multipotential progenitors. These results indicate that the supportive stroma cells possess both secreted and surface bound molecules that protect multipotentiality of HSCs. Global gene expression analysis revealed that the supportive stroma lines from both the placenta and the fetal liver were almost identical (r=0.99) and very different from the non-supportive lines that promote differentiation (r=0.34), implying that they represent two distinct niche cell types. Interestingly, the non-supportive lines express known mesenchymal markers such as (CD73, CD44 and CD166), whereas the identity of the supportive cells is less obvious. In summary, we have identified unique human stromal niche cells that may be critical components of the HSC niches in the placenta and the fetal liver. Molecular characterization of these stroma lines may enable us to define key mechanisms that govern the multipotentiality of HSCs.

Differentiation Profiling of Marrow Stem Cells: A Megakaryocytic Hotspot and the Continuum Model of Hematopoiesis

Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. Standard hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show here that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. This along with other data strongly suggest that stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum and believe that sensitivity to cytokines change as a stem/progenitor cells goes through cell cycle transit. We previously have shown that stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Further work has shown that adhesion protein, cytokine receptor, gene expression and progenitor phenotypes also shift. Evolving data indicate the phenotype of murine marrow stem cells reversible change with cell cycle transit. Murine experiments have been performed on highly purified quiescent G0–1 lineagenegativerhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor, they synchronously pass through cell cycle as measured by propidium iodide, cell doublings and tritiated thymidine. LRH cells enter S-phase in a synchronized fashion by 18 hours, leave S-phase at 40–42 hours and divide between 44–48 hours. The capacity of these cells to respond to a differentiation inductive signal (granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and steel factor) is altered at different points in cell cycle. We have demonstrated differentiation hotspots on a cell cycle continuum (Exp Heme 35:96, 2007). In this work we showed marked but reversible increases in differentiation potential to megakryocyte and granulocytes at different phases of a single cytokine induced cell cycle passage of highly purified quiescent murine LRH marrow stem cells. We have reproducibly induced directed stem cell differentiation by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. We have found that using a differentiation cytokine cocktail of G-CSF at 0.075ng/ml, GM-CSF at 0.0375ng/ml and steel factor at 50ng/ml, we were able to see enhanced megakaryopoiesis occurring 14-days after culture in those LRH stem cells that were in early to mid S-phase at time of inductive signaling. We have now shown that a megakaryocyte hotspot clusters around giving an inductive signal after 32-hours in primary culture; the G1/S interface, and that dramatic reversible changes in differentiation potential occur over half hour time intervals. We have confirmed this data by looking at LRH cells through cell cycle transit after initial cell division showing that a megakaryocyte hotspot occurs in two sequential cell cycles and still tied to S-phase at time of inductive signaling of the daughter cells. This hotspot has been demonstrated on a clonal basis, although the kinetics of the hotspot shifts when clonal as opposed to population studies are carried out. An important issue is whether in vitro cytokine exposure, separate from cell cycle status, determines the existence of the hotspot. To address this, we used Hoechst 33342 dye content to assist in separation of different cell cycle fractions (G0–1, early, mid and late components of S, G2/M) of lineage negative Sca-1+ stem cells, a cycling stem/progenitor cell population in which approximately 20% of the cells are in S-phase at isolation. These cells were only exposed to the differentiation cytokines and showed a megakaryocyte hotspot present in only early S-phase cells after 14-days of culture, showing that in vitro cell cycle phase determined the presence of the hotspot, separate from cytokine exposure. These data indicate that differentiation potential of marrow stem cells exists on a cell cycle related continuum and that this potential can be demonstrated on a single cell basis. Stem cell differentiation hotspots may eventually be utilized to alter repopulation kinetics after bone marrow transplantation improving recovery time of platelets and neutrophils, translating into improved outcomes.

Dorsomorphin, a Selective Small Molecule Inhibitor of BMP Signaling, Promotes Cardiomyogenesis in Embryonic Stem Cells

BackgroundPluripotent embryonic stem (ES) cells, which have the capacity to give rise to all tissue types in the body, show great promise as a versatile source of cells for regenerative therapy. However, the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown, and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules, particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade, hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types.Methodology/ Principal FindingsWe describe the use of dorsomorphin, a selective small molecule inhibitor of BMP signaling, to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust, increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin, unlike the endogenous BMP antagonist Noggin, robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage, but reduces the differentiation of endothelial, smooth muscle, and hematopoietic cells.Conclusions/ SignificanceAdministration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage, apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine.

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.

The effect of silica nanoparticle-modified surfaces on cell morphology, cytoskeletal organization and function

Chemical and morphological characteristics of a biomaterial surface are thought to play an important role in determining cellular differentiation and apoptosis. In this report, we investigate the effect of nanoparticle (NP) assemblies arranged on a flat substrate on cytoskeletal organization, proliferation and metabolic activity on two cell types, Bovine aortic endothelial cells (BAECs) and mouse calvarial preosteoblasts (MC3T3-E1). To vary roughness without altering chemistry, glass substrates were coated with monodispersed silica nanoparticles of 50, 100 and 300 nm in diameter. The impact of surface roughness at the nanoscale on cell morphology was studied by quantifying cell spreading, shape, cytoskeletal F-actin alignment, and recruitment of focal adhesion complexes (FAC) using image analysis. Metabolic activity was followed using a thiazolyl blue tetrazolium bromide assay. In the two cell types tested, surface roughness introduced by nanoparticles had cell type specific effects on cell morphology and metabolism. While BAEC on NP-modified substrates exhibited smaller cell areas and fewer focal adhesion complexes compared to BAEC grown on glass, MC3T3-E1 cells in contrast exhibited larger cell areas on NP-modified surfaces and an increased number of FACs, in comparison to unmodified glass. However, both cell types on 50 nm NP had the highest proliferation rates (comparable to glass control) whereas cells grown on 300 nm NP exhibited inhibited proliferation. Interestingly, for both cell types surface roughness promoted the formation of long, thick F-actin fibers, which aligned with the long axis of each cell. These findings are consistent with our earlier result that osteogenic differentiation of human mesenchymal progenitor cells is enhanced on NP-modified surfaces. Our finding that nanoroughness, as imparted by nanoparticle assemblies, effects cellular processes in a cell specific manner, can have far reaching consequences on the development of “smart” biomaterials especially for directing stem cell differentiation.

Pax3 activation promotes the differentiation of mesenchymal stem cells toward the myogenic lineage

Mesenchymal stem cells (MSCs) residing within the bone marrow (BM) differentiate into multiple lineages, including fat, bone, and cartilage. Because MSCs are multipotent and have a great capacity to be expanded in vitro, these cells are an attractive candidate for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, application of MSCs to muscle degenerative diseases has been hampered by the poor differentiation of MSCs into the muscle lineage. To date most methods require the presence of strong non-physiological agents, such as azacytidine. In the present study we explored the potential of Pax3, the master regulator of the embryonic myogenic program, to promote myogenic differentiation from MSCs. Our results clearly demonstrate that Pax3 promotes the differentiation of MSCs towards the myogenic lineage, which occurs at the expense of other mesenchymal lineages including fat, bone, and cartilage. This effect is cell type-selective since Pax3 overexpression in endothelial cells fails to promote myogenesis. These results highlight the potential of regulating transcriptional pathways to direct differentiation of adult stem cells.

Highly enriched cardiomyocytes from human embryonic stem cells

Current efforts to direct differentiation of human embryonic stem cells (hESC) into a particular cell lineage usually lead to a heterogeneous cell population with only a fraction of the desired cell type present. We show the generation of an essentially pure population of human cardiomyocytes from hESC using lineage selection. A construct comprising the murine alpha-myosin heavy chain (alpha-MHC) promoter driving the neomycin-resistance gene was introduced into hES3 cells to generate stable transgenic lines. Transgenic hESC lines were differentiated into cardiomyocytes and subjected to G418 selection. Both G418-selected and non-selected cardiomyocytes were characterized by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The teratoma-forming potential of differentiated cells was assessed by injection of about 2 million cells into the hind limb muscle of SCID mice. Results After cardiac differentiation and antibiotic selection in a suspension culture process, more than 99% of the transgenic cells showed immunoreactivity to alpha-MHC and alpha-actinin; this enrichment efficiency was observed for independent transgenic cell lines. Quantitative RT-PCR analysis revealed high levels of enrichment for cardiac-specific messages in the selected population. Importantly, injection of selected cells into six SCID mice resulted in no apparent teratoma formation, in contrast to differentiated but non-selected controls. Our results represent a significant step toward scalable production of pure human cardiomyocytes from stable, expandable hESC lines that will facilitate the development of cell therapies, safety pharmacology and drug discovery.