Human adult skeletal muscle stem cells differentiate into cardiomyocyte phenotype in vitro

Cell transplantation to repair or regenerate injured myocardium is a new frontier in the treatment of cardiovascular disease. Most studies on stem cell transplantation therapy in both experimental heart infarct and in phase-I human clinical trials have focused on the use of undifferentiated stem cells. Based on our previous observations demonstrating the presence of multipotent progenitor cells in human adult skeletal muscle, in this study we investigated the capacity of these progenitors to differentiate into cardiomyocytes. Here we show an efficient protocol for the cardiomyogenic differentiation of human adult skeletal muscle stem cells in vitro. We found that treatment with Retinoic Acid directed cardiomyogenic differentiation of skeletal muscle stem cells in vitro. After Retinoic Acid treatment, cells expressed cardiomyocyte markers and acquired spontaneous contraction. Functional assays exhibited cardiac-like response to increased extracellular calcium. When cocultured with mouse cardiomyocytes, Retinoic Acid-treated skeletal muscle stem cells expressed connexin43 and when transplanted into ischemic heart were detectable even 5 weeks after injection. Based on these results, we can conclude that human adult skeletal muscle stem cells, if opportunely treated, can transdifferentiate into cells of cardiac lineage and once injected into infarcted heart can integrate, survive in cardiac tissue and improve the cardiac function.     

Skin-derived human adult stem cells surprisingly share many features with human pancreatic stem cells

Multiple tissue niches in the human body are now recognised to harbour stem cells. Here, we have asked how different adult stem cell populations, isolated from two ontogenetically distinct human organs (skin, pancreas), actually are with respect to a panel of standard markers/characteristics. Here we show that an easily accessible adult human tissue such as skin may serve as a convenient source of adult stem cell-like populations that share markers with stem cells derived from an internal, exocrine organ. Surprisingly, both, human pancreas- and skin-derived stem/progenitor cells demonstrate differentiation patterns across lineage boundaries into cell types of ectoderm (e.g. PGP 9.5+ and GFAP+), mesoderm (e.g. alpha-SMA+) and entoderm (e.g. amylase+ and albumin+). This intriguing differentiation capability warrants systemic follow-up, since it raises the theoretical possibility that an adult human skin-derived progenitor cell population could be envisioned for possible application in cell replacement therapies.     

Efficient in vitro myogenic reprogramming of human primary mesenchymal stem cells and endothelial cells by Myf5

Background information. The identification of a source of stem cells able to regenerate skeletal muscle was the goal of numerous studies with the aim to develop new therapeutic approaches for genetic muscle diseases or muscle injuries. A series of studies have demonstrated that stem cells derived from various tissues may have a role in the regeneration of damaged muscles, but this contribution is always very weak. Thus we established a project aiming to reprogramme non‐muscle cells into the skeletal striated differentiation pathway. Results. We transduced several human primary adult stem or progenitor cells using a recombinant lentivirus containing the coding sequence of the Myf5 gene considered as a master gene for the determination of skeletal striated muscle. These original results are the first demonstration of a myogenic conversion of human mesenchymal and endothelial cells by Myf5. Conclusions. The procedure described in the present paper could be used to develop new research protocols with the prospect of using these cells as therapeutic agents.     

Conditional TGF‐β1 treatment increases stem cell‐like cell population in myoblasts

The limitation in successfully acquiring large populations of stem cell has impeded their application. A new method based on the dedifferentiation of adult somatic cells to generate induced multipotent stem cells would allow us to obtain a large amount of autologous stem cells for regenerative medicine. The current work was proposed to induce a sub‐population of cells with characteristics of muscle stem cells from myoblasts through conditional treatment of transforming growth factor (TGF)‐β₁. Our results show that a lower concentration of TGF‐β₁ is able to promote C2C12 myoblasts to express stem cell markers as well as to repress myogenic proteins, which involves a mechanism of dedifferentiation. Moreover, TGF‐β₁ treatment promoted the proliferation‐arrested C2C12 myoblasts to re‐enter the S‐phase. We also investigated the multi‐differentiation potentials of the dedifferentiated cells. TGF‐β₁ pre‐treated C2C12 myoblasts were implanted into mice to repair dystrophic skeletal muscle or injured bone. In addition to the C2C12 myoblasts, similar effects of TGF‐β₁ were also observed in the primary myoblasts of mice. Our results suggest that TGF‐β₁ is effective as a molecular trigger for the dedifferentiation of skeletal muscle myoblasts and could be used to generate a large pool of progenitor cells that collectively behave as multipotent stem cell‐like cells for regenerative medicine applications.     

Host tissue response in stem cell therapy

Preclinical and clinical trials of stem cell therapy have been carried out for treating a broad spectrum of diseases using several types of adult stem cells. While encouraging therapeutic results have been obtained, much remains to be investigated regarding the best cell type to use, cell dosage, delivery route, long-term safety, clinical feasibility, and ultimately treatment cost. Logistic aspects of stem cell therapeutics remain an area that requires urgent attention from the medical community. Recent cardiovascular trial studies have demonstrated that growth factors and cytokines derived from the injected stem cells and host tissue appear to contribute largely to the observed therapeutic benefits, indicating that trophic actions rather than the multilineage potential (or stemness) of the administered stem cells may provide the underlying tissue healing power. However, the capacity for trophic factor production can be aberrantly downregulated as seen in human heart disease. Skeletal muscle is a dynamic tissue with an impressive ability to continuously respond to environmental stimuli. Indeed, a relation exists between active skeletal muscle and low cardiovascular risk, highlighting the critical link between the skeletal muscle and cardiovascular systems. Adding to this notion are recent studies showing that stem cells injected into skeletal muscle can rescue the failing rodent heart through activation of the muscle trophic factor network and mobilization of bone marrow multilineage progenitor cells. However, aging and disease can adversely affect the host tissue into which stem cells are injected. A better understanding of the host tissue response in stem cell therapy is necessary to advance the field and bridge the gap between preclinical and clinical findings.     

A perivascular origin for mesenchymal stem cells in multiple human organs

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rbeta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.     

Generation of different fates from multipotent muscle stem cells

Although neuronal and mesenchymal stem cells exhibit multipotentiality, this property has not previously been demonstrated for muscle stem cells. We now show that muscle satellite cells of adult mice are able to differentiate into osteoblasts, adipocytes and myotubes. Undifferentiated muscle progenitor cells derived from a single satellite cell co-expressed multiple determination genes including those for MyoD and Runx2, which are specific for myogenic and osteogenic differentiation, respectively. Determination genes not relevant to the induced differentiation pathway were specifically downregulated in these cells. Similar multipotent progenitor cells were isolated from adult human muscle. Based on these observations, we propose a 'stock options' model for the generation of different fates from multipotent stem cells.     

Harnessing the therapeutic potential of myogenic stem cells

The potential clinical use of stem cells for cell transplantation therapies to replace defective genes in myopathies is an area of intense investigation. Precursor cells derived from non-muscle tissue with myogenic potential have been identified in many tissues, including bone marrow and dermis, although the status of these putative stem cells requires clarification. The incorporation of circulating bone-marrow derived stem cells into regenerating adult skeletal muscle has been demonstrated in mice but the contribution of donor cells is so minimal that it would appear clinically irrelevant at this stage. The possibility of a true stem cell subpopulation within skeletal muscle that replenishes the satellite cells (conventional muscle precursors on the surface of myofibres) is also very attractive as a superior source of myoblasts for muscle construction. A full understanding of the intrinsic factors (i.e. gene expression within the stem cell) and extrinsic factors (i.e. signals from the external environment) which control the commitment of stem cells to the myogenic lineage, and the conditions which favour stem cell expansion in vivo is required before stem cells can be seriously considered for clinical cell therapy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.     

Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantify the hematopoietic stem cell potential, we have used highly purified SP/CD45^＋ cells in long-term culture initiating cell （LTC-IC） assays. The SP/CD45^＋ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45^＋ cells from muscle exhibited robust proliferative activity in vitro at day 16 （380-fold amplification）, especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.     

Extracellular matrix components direct porcine muscle stem cell behavior

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.     

A modified enrichment protocol for adult caprine skeletal muscle stem cell

To establish an adequate model to study the proliferation and differentiation of adult caprine skeletal muscle in response to bioactive compounds, a pool of satellite cells (SC) was derived from the rectus abdominis muscle of adult goat. Skeletal muscle contains a population of adult stem cells, named as satellite cells that reside beneath the basal lamina of skeletal muscle fiber and other populations of cells. These SC are multipotent stem cells, since cells cultured in the presence of specific cell lineage inducing cocktails can differentiate into several types of mesenchymal lineage, such as osteocytes and adipocytes. In the present study, we have developed a modified protocol for isolating satellite cells (>90%) and examined their myogenic and contractile properties in vitro.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Programming human pluripotent stem cells into white and brown adipocytes

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.     

Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.     

胚胎干细胞向心肌细胞分化过程中能量代谢变化的研究

目的：定向诱导人胚胎干细胞分化为心肌细胞，对分化过程中胚胎干细胞、心肌祖细胞和心肌细胞糖酵解能力和线粒体氧化磷酸化能力进行实时定量检测，探索分化过程中细胞能量代谢表型的转换机制.方法：用GSK3抑制剂CHIR99021和Wnt信号通路小分子抑制剂IWP2的方法定向分化人胚胎干细胞为心肌祖细胞和心肌细胞；细胞免疫荧光检测人胚胎干细胞标志物，流式细胞术检测人心肌祖细胞和心肌细胞标志物；应用细胞外流量分析（Extracellular Flux Analysis）方法检测人胚胎干细胞、心肌祖细胞和心肌细胞能量代谢情况.结果：人胚胎干细胞干性保持稳定，均表达Nanog、OCT4、SOX2细胞标志物；在向心肌分化过程中，第7d心肌祖细胞标志物Isl1表达99%以上，分化第15d心肌细胞标志物cTnT表达83%以上；人胚胎干细胞糖酵解代谢能力最强，心肌细胞线粒体功能最强，心肌祖细胞处于两种代谢方式的过度阶段.结论：在人胚胎干细胞向心肌细胞分化的过程中，细胞糖酵解能力逐渐减弱，线粒体氧化磷酸化能力逐渐增强，细胞的能量代谢类型发生转变.     

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoD^iCre/+;R26R^EYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate.