肌源干细胞可塑性研究进展

目前已证实肌肉中至少存在两种干细胞:肌卫星细胞和肌源干细胞。肌源干细胞被认为是卫星细胞的前体细胞,具有较高的增殖能力、更好的细胞生存能力和更宽的分化能力。肌源干细胞不仅能够分化成血、肌肉、脂肪、骨、软骨、内皮等中胚层细胞,而且也能打破胚层限制分化成外胚层和内胚层细胞。文章对肌源干细胞的分离纯化、鉴定、可塑性及临床应用做一综述。     

Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.     

Isolation and passage of muscle-derived stem cells from the rat penile corpora cavernosa and induction of differentiation into smooth muscle cells

This study treated the isolation and passage of muscle-derived stem cells (MDSCs) from rat penile corpora cavernosa, detection of stem cell marker expression, observation of their self-renewal and continuous proliferation, and demonstration of their potential to differentiate into smooth muscle cells in co-culture. Muscle-derived stem cells from the rat penile corpora cavernosa were isolated and purified. The expression of stem cell markers Sca-1 and desmin was detected in PP6 cells, thus confirming that the main components of PP6 cells are MDSCs. The expression of Sca-1 and desmin occurred both in PP6 cells and cells at passages 3, 6, and 8, and there was no significant decrease in the expression level with increasing passage number. The growth curves indicated that the cell doubling time was approximately 48 h. The cells entered the stationary phase after approximately 7 days of culture. The proliferative activity of the cells at passage 8 remained unchanged. After 2 days of co-culture with smooth muscle cells, the DAPI-labeled MDSCs tended to exhibit smooth muscle cell morphology and expression of α-SMA was detected. MDSCs exist in the rat penile corpora cavernosa and possess the potential to differentiate into smooth muscle cells. This discovery serves as the basis in view of the potential use of endogenous stem cells for the treatment of erectile dysfunction (ED).     

Long-term self-renewal of postnatal muscle-derived stem cells

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.     

肌源干细胞研究进展

目前已证实肌肉是具有多向分化潜能的成体干细胞的一个储存库。研究者认为骨骼肌中至少有两种干细胞：肌卫星细胞（muscle satellite cells）和肌源干细胞（muscle-derived stem cells, MDSCs）,并且使用几种方法从肌肉中分离获得不同类群的MDSCs。研究发现分离这些细胞的方法影响干细胞的特征。本文对MDSCs的行为、生物学特征、分离、分化及其在治疗组织器官修复和再生中应用的可能性等作一概括介绍。     

Tissue engineering with muscle-derived stem cells

Tissue engineering and cell therapy approaches aim to take advantage of the repopulating ability and plasticity of multipotent stem cells to regenerate lost or diseased tissue. Researchers continue to investigate stem cells in mature tissues and demonstrate the potential ability of organ-specific cells to differentiate into multiple lineages. One stem cell that displays such promise is the muscle-derived stem cell (MDSC). Data supporting the existence of MDSCs have emerged as part of investigations to improve myoblast cell transplantation for the treatment of muscular dystrophies. As these efforts continue, the potential for MDSC-based therapy for other musculoskeletal injuries, as well as for cardiac and smooth muscle injuries, is currently being explored.     

Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration

Three populations of myogenic cells were isolated from normal mouse skeletal muscle based on their adhesion characteristics and proliferation behaviors. Although two of these populations displayed satellite cell characteristics, a third population of long-time proliferating cells expressing hematopoietic stem cell markers was also identified. This third population comprises cells that retain their phenotype for more than 30 passages with normal karyotype and can differentiate into muscle, neural, and endothelial lineages both in vitro and in vivo. In contrast to the other two populations of myogenic cells, the transplantation of the long-time proliferating cells improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle. The long-time proliferating cells' ability to proliferate in vivo for an extended period of time, combined with their strong capacity for self-renewal, their multipotent differentiation, and their immune-privileged behavior, reveals, at least in part, the basis for the improvement of cell transplantation. Our results suggest that this novel population of muscle-derived stem cells will significantly improve muscle cell-mediated therapies.     

alpha7 integrin expressing human fetal myogenic progenitors have stem cell-like properties and are capable of osteogenic differentiation

During muscle development, precursor cells fuse to form myofibers. Following injury in adult muscle, quiescent satellite cells become activated to regenerate muscle in a fashion similar to fetal development. Recent studies indicate that murine skeletal myoblasts can differentiate along multiple cell lineages including the osteoblastic pathway. However, little is known about the multipotency of human myogenic cells. Here, we isolate myogenic precursor cells from human fetal and adult muscle by sorting for the laminin-binding alpha7 integrin and demonstrate their differentiation potential and alteration in adhesive behavior. The alpha7-positive human fetal progenitors were efficient at forming myotubes and a majority expressed known muscle markers including M-cadherin and c-Met, but were heterogeneous for desmin and MyoD expression. To test their pluripotent differentiation potential, enriched populations of alpha7-positive fetal cells were subjected to inductive protocols. Although the myoblasts appeared committed to a muscle lineage, they could be converted to differentiate along the osteoblastic pathway in the presence of BMP-2. Interestingly, osteogenic cells showed altered adhesion and migratory activity that reflected growth factor-induced changes in integrin expression. These results indicate that alpha7-expressing fetal myoblasts are capable of differentiation to osteoblast lineage with a coordinated switch in integrin profiles and may represent a mechanism that promotes homing and recruitment of myogenic stem cells for tissue repair and remodeling.     

CD117+ amniotic fluid stem cells: State of the art and future perspectives

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

Mesenchymal stem cells: Molecular characteristics and clinical applications

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells with the capacity to differentiate into tissues of both mesenchymal and non-mesenchymal origin. MSCs can differentiate into osteoblastic, chondrogenic, and adipogenic lineages, although recent studies have demonstrated that MSCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages. Since their original isolation from the bone marrow, MSCs have been successfully harvested from many other tissues. Their ease of isolation and ex vivo expansion combined with their immunoprivileged nature has made these cells popular candidates for stem cell therapies. These cells have the potential to alter disease pathophysiology through many modalities including cytokine secretion, capacity to differentiate along various lineages, immune modulation and direct cell-cell interaction with diseased tissue. Here we first review basic features of MSC biology including MSC characteristics in culture, homing mechanisms, differentiation capabilities and immune modulation. We then highlight some in vivo and clinical evidence supporting the therapeutic roles of MSCs and their uses in orthopedic, autoimmune, and ischemic disorders.     

CD117+ amniotic fluid stem cells

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

Myogenic and neurogenic differentiation of human tooth germ stem cells (hTGSCs) are regulated by pluronic block copolymers

Stem cells with high proliferation, self-renewal and differentiation capacities are promising for tissue engineering approaches. Among stem cells, human tooth germ stem cells (hTGSCs) having mesenchymal stem cell characteristics are highly proliferative and able to differentiate into several cell lineages. Researchers have recently focused on transplanting stem cells with bioconductive and/or bioinductive materials that can provide cell commitment to the desired cell lineages. In the present study, effects of pluronic block copolymers (F68, F127 and P85) on in vitro myo- and neurogenic differentiation of human tooth germ stem cells (hTGSCs) were investigated. As P85 was found to exert considerable toxicity to hTGSCs even at low concentrations, it was not evaluated for further differentiation experiments. Immunocytochemical analysis, gene and protein expression studies revealed that while F68 treatment increased lineage-specific gene expression in both myo- and neuro-genically differentiated cells, F127 did not result in any remarkable difference compared to cells treated with differentiation medium. Subsequent studies are required to explore the exact mechanisms of how F68 increases the myogenic and neurogenic differentiation of hTGSCs. The present work indicates that pluronic F68 might be used in functional skeletal and neural tissue engineering applications.     

Contribution of retinoid X receptor signaling to the specification of skeletal muscle lineage

Pluripotent stem cells possess a tremendous potential for the treatment of many diseases because of their capacity to differentiate into a variety of cell lineages. However, they provide little promise for muscle-related diseases, mainly because of the lack of small molecule inducers to efficiently direct myogenic conversion. Retinoic acid, acting through the retinoic acid receptor (RAR) and retinoid X receptor (RXR), affects stem cell fate determination in a concentration-dependent manner, but it only has a modest efficacy on the commitment of ES cells into skeletal muscle lineage. The RXR is very important for embryonic development but is generally considered to act as a silent partner of RAR in a non-permissive mode. In this study, we have examined whether activation of the RXR by rexinoid or RXR-specific signaling play a role in the specification of stem cells into muscle lineage. Our findings demonstrate that mouse ES cells generate skeletal myocytes effectively upon treatment with rexinoid at the early stage of differentiation and that on a molecular level, rexinoid-enhanced myogenesis simulates the sequential events observed in vivo. Moreover, RXR-mediated myogenic conversion requires the function of β-catenin but not RAR. Our studies establish the feasibility of applying the RXR agonist in cell-based therapies to treat muscle-related diseases. The aptitude of mouse ES cells to generate skeletal myocytes following rexinoid induction also provides a model system to study the convergence of different signaling pathways in myogenesis.     

Muscle origin of porcine satellite cells affects in vitro differentiation potential

Post‐natal muscle regeneration relies on the activation of tissue stem cells known as satellite cells, to repair damage following exercise trauma and disease. Satellite cells from individual muscles are known to be heterogeneous with regard to proliferation, fusion and transplantation abilities, although the muscle origin has rarely been considered pertinent to their differentiation capabilities. In this study we compared the potential of two functionally distinct skeletal muscle satellite cell populations from porcine diaphragm and hind‐limb semi‐membranosus muscles. These two muscles were chosen primarily for differences in metabolic and contractile properties: the diaphragm is more continuously active and has a greater oxidative capacity. Cells were induced to differentiate towards myogenic and adipogenic lineages, and here we have shown that cells from diaphragm exhibit a significantly greater degree of myogenesis compared with those from semi‐membranosus, while the converse was true for adipogenesis. Unexpectedly, both conditions generated small numbers of cells with neuronal characteristics for both muscle types, although more so in cells derived from the diaphragm. With increased interest in muscle adiposity with age and disease, these findings suggest that muscle origin of satellite cells does affect lineage fate, however whether differences in developmental origin or metabolic activity of the parent tissue govern this, remains to be determined. Copyright © 2010 John Wiley & Sons, Ltd.     

The potential of muscle stem cells.

Skeletal muscle contains two types of stem cells: satellite cells, which function as myogenic precursors, and a population of multipotent adult stem cells. Satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. An additional stem cell population in adult muscle displays a remarkable capacity to differentiate into hematopoietic cells as well as muscle following transplantation. This article discusses the characteristics and properties of these cell populations, the relationship between them, and the potential for stem cell-based muscle therapeutics.     

Engraftment of mesenchymal stem cells into dystrophin-deficient mice is not accompanied by functional recovery

Mesenchymal stem cell preparations have been proposed for muscle regeneration in musculoskeletal disorders. Although MSCs have great in vitro expansion potential and possess the ability to differentiate into several mesenchymal lineages, myogenesis has proven to be much more difficult to induce. We have recently demonstrated that Pax3, the master regulator of the embryonic myogenic program, enables the in vitro differentiation of a murine mesenchymal stem cell line (MSCB9-Pax3) into myogenic progenitors. Here we show that injection of these cells into cardiotoxin-injured muscles of immunodeficient mice leads to the development of muscle tumors, resembling rhabdomyosarcomas. We then extended these studies to primary human mesenchymal stem cells (hMSCs) isolated from bone marrow. Upon genetic modification with a lentiviral vector encoding PAX3, hMSCs activated the myogenic program as demonstrated by expression of myogenic regulatory factors. Upon transplantation, the PAX3-modified MSCs did not generate rhabdomyosarcomas but rather, resulted in donor-derived myofibers. These were found at higher frequency in PAX3-transduced hMSCs than in mock-transduced MSCs. Nonetheless, neither engraftment of PAX3-modified or unmodified MSCs resulted in improved contractility. Thus these findings suggest that limitations remain to be overcome before MSC preparations result in effective treatment for muscular dystrophies.     

Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications.     

Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8(+) cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.