Gene therapy and tissue engineering based on muscle-derived stem cells

Skeletal muscle represents a convenient source of stem cells for cell-based tissue and genetic engineering. Muscle-derived stem cells (MDSCs) exhibit both multipotentiality and self-renewal capabilities, and are considered to be distinct from the well-studied satellite cell, another type of muscle stem cell that is capable of self-renewal and myogenic lineage differentiation. The MDSC appears to have less restricted differentiation capabilities as compared with the satellite cell, and may be a precursor of the satellite cell. This review considers the evidence for the existence of MDSCs as well as their origin. We will discuss recent investigations highlighting the potential of stem cell transplantation for the treatment of skeletal, cardiac and smooth muscle injuries and disease. We will highlight challenges in bridging the gap between understanding basic stem cell biology and clinical utilization for cell therapy.     

Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics

In an attempt to determine whether muscle-derived stem cells are distinct from satellite cells, we investigated whether muscle-derived stem cells could be isolated from the skeletal muscle of Pax7-deficient mice, which have been shown to be devoid of or to contain only a minimal number of satellite cells. Utilizing a technique that separates cells based on their adhesion characteristics (the preplate technique), several distinct populations of muscle-derived cells were isolated. In these mice, the Pax7 gene was knocked out with the insertion of the LacZ gene. One population was both rapidly adhering, LacZ-positive, and displayed a high myogenic index, but was rapidly lost to terminal differentiation when continuously replated. A second population, which persisted over 50 passages, was LacZ-negative and displayed a low myogenic index. Although Pax3 may have acted as a compensatory mechanism for the myogenic commitment of the LacZ-positive cells, the LacZ-negative cells, despite expressing Pax3, required Pax7 transduction to restore their myogenic capacity. We believe that these two populations of myogenic progenitor cells, each endowed with different adhesion characteristics, may help explain the discrepancy in the literature concerning the presence of myogenic cells found in Pax7-deficient mice.     

The role of α-smooth muscle actin in myogenic differentiation of human glandular stem cells and their potential for smooth muscle cell replacement therapies

Importance of the field: Cellular replacement therapies in vascular and urogenital organ disorders require an abundant source of smooth muscle cells. A promising approach would be the directed myogenic differentiation (characterized by the expression of α-smooth muscle actin (α-SMA)) into a sufficient amount of smooth muscle cells through easily obtainable adult stem cells, for example from the sweat gland.

Areas covered in this review: We present novel multipotent adult stem cell populations derived from glandular tissues like pancreas, salivary gland and sweat gland and assess their myogenic potential. Their possible application in cell replacement therapies is discussed, with regard to numerous scaffold-based approaches in the course of the last decade.

What the reader will gain: Multipotent glandular stem cells can be manipulated by different means to express a predominant smooth muscle-like phenotype. Possible promising applications of myogenic differentiated stem cells were evaluated, since several studies revealed the beneficial effect of somatic stem cells in replacement therapies for blood vessels, bladder reconstructions, etc.

Take home message: Glandular stem cells, especially sweat-gland-derived cells, provide an easily accessible and efficient source for autologous smooth muscle tissue, which might be used to replace vascular tissue in case of organ failure or disorder.     

Myoblasts Derived From Normal hESCs and Dystrophic hiPSCs Efficiently Fuse With Existing Muscle Fibers Following Transplantation

Human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have an endless self-renewal capacity and can theoretically differentiate into all types of lineages. They thus represent an unlimited source of cells for therapies of regenerative diseases, such as Duchenne muscular dystrophy (DMD), and for tissue repair in specific medical fields. However, at the moment, the low number of efficient specific lineage differentiation protocols compromises their use in regenerative medicine. We developed a two-step procedure to differentiate hESCs and dystrophic hiPSCs in myogenic cells. The first step was a culture in a myogenic medium and the second step an infection with an adenovirus expressing the myogenic master gene MyoD. Following infection, the cells expressed several myogenic markers and formed abundant multinucleated myotubes in vitro. When transplanted in the muscle of Rag/mdx mice, these cells participated in muscle regeneration by fusing very well with existing muscle fibers. Our findings provide an effective method that will permit to use hESCs or hiPSCs for preclinical studies in muscle repair.     

Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells

Skeletal muscle damaged by injury or by degenerative diseases such as muscular dystrophy is able to regenerate new muscle fibers. Regeneration mainly depends upon satellite cells, myogenic progenitors localized between the basal lamina and the muscle fiber membrane. However, other cell types outside the basal lamina, such as pericytes, also have myogenic potency. Here, we discuss the main properties of satellite cells and other myogenic progenitors as well as recent efforts to obtain myogenic cells from pluripotent stem cells for patient-tailored cell therapy. Clinical trials utilizing these cells to treat muscular dystrophies, heart failure, and stress urinary incontinence are also briefly outlined.     

In vivo evaluation of human dental pulp stem cells differentiated towards multiple lineages

An increasing number of investigations supports that adult stem cells have the potential to differentiate into matured cell types beyond their origin, a property defined as plasticity. Previously, the plasticity of stem cells derived from dental pulp (DPSC) has been confirmed by culturing cells in lineage-specific media in vitro. In the current study, the in vivo differentiation or maturation potential of DPSC was further analysed, by transplanting human DPSC/collagen scaffold constructs into subcutaneous tissue of immunocompromised mice. Cells received odontogenic, adipogenic or myogenic pre-induction, whereas control samples received no stimulation. Also blank collagen scaffolds were implanted. The results indicated that seeded cells produced tissue within the implanted constructs after 3 weeks of implantation. According to morphological and phenotypical changes, the pre-induced DPSC showed the ability to further differentiate along odontogenic, myogenic and adipogenic pathways in vivo. Moreover, DPSC without pre-treatment were able to spontaneously differentiate along odontogenic and adipogenic directions in vivo. However, only limited mature morphological changes were detected in histology. In summary, stem cells derived from human dental pulp form a suitable source for tissue engineering and cell-mediated therapy, although additional analyses should be considered.     

Cells that participate in regeneration of skeletal muscle

Skeletal muscle is a post-mitotic tissue that is thought, conventionally to be maintained by repair and regeneration by a population of stem cell-like satellite cells. Recent findings have brought into question the extent to which these satellite cells represent a single homogeneous population and whether they are the only source of myogenic cells in mature muscle. It has been shown that myogenic cells can be derived from the bone marrow or from the supposedly post-mitotic nuclei of muscle fibres. However, neither of these sources has been demonstrated to generate more than a tiny proportion of muscle nuclei. It remains possible nonetheless that such mechanisms might be exploited for therapeutic uses in primary muscle disease.     

Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications.     

Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells.

Myoblast transplantation has been investigated as a therapy for muscle-related diseases and as a gene delivery vehicle for therapeutic recombinant proteins. Clinical successes involving muscle cell transplantation have been limited, in part because of poor donor cell survival, and the heterogeneous nature of myogenic donor cells has largely been ignored. We have previously reported an isolation technique, preplating, that results in purified myogenic cells that are capable of significantly higher rates of donor cell survival leading to enhanced gene transfer to skeletal muscle. Characterization of these purified cells revealed that they display markers common to stem cells and are capable of multilineage differentiation. This study was performed to phenotypically characterize, by flow cytometry, muscle-derived cell populations obtained by the preplate technique for the purpose of eventually developing a method to quickly identify and isolate viable muscle cells best suited for transplantation. Muscle cell cultures were analyzed for expression of the surface proteins Sca-1, c-Kit, and CD34. We found that the preplate technique purifies distinct myogenic cell subpopulations expressing CD34 alone (Sca-1 negative) and Sca-1 alone (CD34 negative), but that this expression is subject to change with time in culture. Isolation and transplantation of phenotypically pure Sca-1-positive myogenic cells, obtained by magnetic cell sorting, demonstrates the ability to quickly select viable myogenic cells capable of regenerating skeletal muscle and restoring dystrophin expression within dystrophic host skeletal muscle. Flow cytometric described phenotypes will aid in the rapid isolation of specific donor cell populations for muscle cell transplants and muscle cell-mediated gene therapies, thereby enhancing their future success.     

CD133+ cells isolated from various sources and their role in future clinical perspectives

Background. CD133 is a member of a novel family of cell surface glycoproteins. Initially, the expression of CD133 antigen was seen only in the hematopoietic derived CD34⁺ stem cells. At present, CD133 expression is demonstrated in undifferentiated epithelium, different types of tumors and myogenic cells. CD133⁺ neurosphere cells isolated from brain are able to differentiate into both neurons and glial cells. These data suggested that CD133 could be a specific marker for various stem and progenitor cell populations.

Objectives. The main goal would be to describe the role for CD133 as a marker of stem cells able to engraft and differentiate, to form functional non-hematopoietic adult lineages and contribute to disease amelioration via tissue regeneration.

Results/conclusion. In conclusion, since the rise of CD133 antigen as a suitable stem cell marker, the possible use of CD133⁺ stem cells in therapeutic applications has opened a new promising field in the treatment of degenerating diseases. The human circulating cells expressing the CD133 antigen behave as a stem cell population capable of commitment to hematopoietic, endothelial and myogenic lineages. CD133 cell therapy may represent a promising treatment for many diseases.     

In Vivo Myogenic Potential of Human CD133+ Muscle-derived Stem Cells: A Quantitative Study

In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle–derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2^−/− γC^−/− C5^−/− mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle–derived CD133⁺ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133⁺/CD34⁺ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133⁺ cells could be an attractive candidate for cellular therapy.     

Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose�Cderived Stem Cells With Forced Expression of MyoD

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose–derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2^−/−γC^−/− mice resulted in a substantial increase in the number of hMADS cell–derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.     

CDK4 and cyclin D1 allow human myogenic cells to recapture growth property without compromising differentiation potential

In vitro culture systems of human myogenic cells contribute greatly to elucidation of the molecular mechanisms underlying terminal myogenic differentiation and symptoms of neuromuscular diseases. However, human myogenic cells have limited ability to proliferate in culture. We have established an improved immortalization protocol for human myogenic cells derived from healthy and diseased muscles; constitutive expression of mutated cyclin-dependent kinase 4, cyclin D1 and telomerase immortalized human myogenic cells. Normal diploid chromosomes were preserved after immortalization. The immortalized human myogenic cells divided as rapidly as primary human myogenic cells during the early passages, and underwent myogenic, osteogenic and adipogenic differentiation under appropriate culture conditions. The immortalized cells contributed to muscle differentiation upon xenotransplantation to immunodeficient mice under conditions of regeneration following muscle injury. We also succeeded in immortalizing cryopreserved human myogenic cells derived from Leigh disease patients following primary culture. Forced expression of the three genes shortened their cell cycle to < 30 h, which is similar to the doubling time of primary cultured human myogenic cells during early passages. The immortalization protocol described here allowed human myogenic cells to recapture high proliferation activity without compromising their differentiation potential and normal diploidy.     

Time‐lapse microscopy and classification of 2D human mesenchymal stem cells based on cell shape picks up myogenic from osteogenic and adipogenic differentiation

Current methods to characterize mesenchymal stem cells (MSCs) are limited to CD marker expression, plastic adherence and their ability to differentiate into adipogenic, osteogenic and chondrogenic precursors. It seems evident that stem cells undergoing differentiation should differ in many aspects, such as morphology and possibly also behaviour; however, such a correlation has not yet been exploited for fate prediction of MSCs. Primary human MSCs from bone marrow were expanded and pelleted to form high‐density cultures and were then randomly divided into four groups to differentiate into adipogenic, osteogenic chondrogenic and myogenic progenitor cells. The cells were expanded as heterogeneous and tracked with time‐lapse microscopy to record cell shape, using phase‐contrast microscopy. The cells were segmented using a custom‐made image‐processing pipeline. Seven morphological features were extracted for each of the segmented cells. Statistical analysis was performed on the seven‐dimensional feature vectors, using a tree‐like classification method. Differentiation of cells was monitored with key marker genes and histology. Cells in differentiation media were expressing the key genes for each of the three pathways after 21 days, i.e. adipogenic, osteogenic and chondrogenic, which was also confirmed by histological staining. Time‐lapse microscopy data were obtained and contained new evidence that two cell shape features, eccentricity and filopodia (= 'fingers') are highly informative to classify myogenic differentiation from all others. However, no robust classifiers could be identified for the other cell differentiation paths. The results suggest that non‐invasive automated time‐lapse microscopy could potentially be used to predict the stem cell fate of hMSCs for clinical application, based on morphology for earlier time‐points. The classification is challenged by cell density, proliferation and possible unknown donor‐specific factors, which affect the performance of morphology‐based approaches. Copyright © 2012 John Wiley & Sons, Ltd.     

胎儿骨髓和肝脏间充质干细胞的表型和生物学性状研究

为研究胎儿骨髓和肝脏间充质干细胞的表型和生物学性状,取胎龄为4-5个月水囊引产胎儿,将骨髓和肝脏细胞在SF（含2?S）培养基中培养,进行电镜观察,测定生长曲线,用流式细胞术对培养细胞进行表型测定。细胞周期分析。用SA方法测定Ⅰ,Ⅲ型胶原和vWF因子表达。结果表明;从胎儿骨髓和肝脏培养出的间充质干细胞,两在形态学,生长特性,免疫表型上是相似的,肝脏间充质干细胞有更好的支持造血的功能。结论提示,从胎儿骨髓和肝脏可分离培养出间充质干细胞。在体外有效扩增且保持其低分化状态。     

Differentiation potential of adult human mesenchymal stem cells

Human mesenchymal stem cells (HMSCs) which are isolated from bone marrow stroma, peripheral blood, dermis, muscle and adipose tissue have the advantage of potential autologous transplantation ability. They can be differentiated into chondrogenic, osteogenic, adipogenic and myogenic lineages. Problems of stem cells from bone marrow are low cell numbers, low isolated volumes, pain, and to some extent ethical concerns. The isolation of mesenchymal stem cells from human adipose tissue was recently identified as an alternative source, since these cells are easy to obtain in big cell numbers. Adipose tissue is derived from embryonic mesoderm and contains a heterogeneous stromal cell population. To achieve lineage-specific differentiation of these cells they have to be cultured in media supplemented with appropriate factors. Inductions of the cells into multiple mesenchymal lineages resulted in the expression of several lineage-specific genes, proteins and specific metabolic activity. In conclusion, the potential benefit of the multi-germline capacity of HMSCs seems to be a promising approach for allogenic cell therapy and human tissue engineering.     

Clinical review: Stem cell therapies for acute lung injury/acute respiratory distress syndrome - hope or hype?

ABSTRACT: A growing understanding of the complexity of the pathophysiology of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), coupled with advances in stem cell biology, has led to a renewed interest in the therapeutic potential of stem cells for this devastating disease. Mesenchymal stem cells appear closest to clinical translation, given the evidence that they may favourably modulate the immune response to reduce lung injury, while maintaining host immune-competence and also facilitating lung regeneration and repair. The demonstration that human mesenchymal stem cells exert benefit in the endotoxin-injured human lung is particularly persuasive. Endothelial progenitor cells also demonstrate promise in reducing endothelial damage, which is a key pathophysiological feature of ALI. Embryonic and induced pluripotent stem cells are at an earlier stage in the translational process, but offer the hope of directly replacing injured lung tissue. The lung itself also contains endogenous stem cells, which may ultimately offer the greatest hope for lung diseases, given their physiologic role in replacing and regenerating native lung tissues. However, significant deficits remain in our knowledge regarding the mechanisms of action of stem cells, their efficacy in relevant pre-clinical models, and their safety, particularly in critically ill patients. These gaps need to be addressed before the enormous therapeutic potential of stem cells for ALI/ARDS can be realised.     

细胞因子修饰的间充质干细胞

背景：将细胞治疗与基因治疗相结合,通过不同的细胞因子对间充质干细胞的修饰,不但可以赋予其新的能力,更能增强其本身的功能。但是在西北高原的低氧环境下,间充质干细胞的功能发挥就会受到一定的限制。目的：文章综述了间充质干细胞近年来的研究进展,在与细胞因子联合应用后功能上的扩展,并寻找新的手段使其在高原低氧环境下充分发挥功能。方法：应用计算机检索CNKI数据库和PUBMED数据库中关于间充质干细胞的文章,以＂间充质干细胞;低氧诱导因子;肝细胞生长因子＂或＂mesenchymal stem cells;hypoxia inducible factor;hepatocyte growth factor＂为检索词进行检索。检索文献总量为71篇,选择文章内容与间充质干细胞及其临床应用有关方面的文章,同一领域文献则选择近期发表或发表在权威杂志文章。结果与结论：最终纳入35篇符合标准的文献。通过应用细胞因子的转染,将细胞因子的功能与间充质干细胞相结合,使得间充质干细胞的功能更全面,在高原低氧的恶劣环境下,依然能够有效的发挥临床效用,满足其临床上的治疗需求。     

Human circulating AC133(+) stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle

Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133(+) cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYF5. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies.