Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro

Background information. DMD (Duchenne muscular dystrophy) is a devastating X‐linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose‐derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X‐linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. Results. We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co‐cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)‐positive ASCs and DAPI (4′,6‐diamidino‐2‐phenylindole)‐stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. Conclusions. These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.     

Mesenchymal stem cells as anti-inflammatories: Implications for treatment of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked musculodegenerative condition consisting of an underlying genetic defect whose manifestation is augmented by inflammatory mechanisms. Previous treatment approaches using gene replacement, exon-skipping or allogeneic cell therapy have been relatively unsuccessful. The only intervention to mediate improvement in survival, albeit minor, is glucocorticoid treatment. Given this modality appears to function via suppression of underlying inflammation; we focus this review on the inflammatory response as a target for mesenchymal stem cell (MSC) therapy. In contrast to other cell based therapies attempted in DMD, MSC have the advantages of (a) ability to fuse with and genetically complement dystrophic muscle; (b) possess anti-inflammatory activities; and (c) produce trophic factors that may augment activity of endogenous repair cells. We conclude by describing one practical scenario of stem cell therapy for DMD.     

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro.

BACKGROUND INFORMATION: DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. RESULTS: We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4',6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. CONCLUSIONS: These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.     

Prospects for pluripotent stem cell‐derived cardiomyocytes in cardiac cell therapy and as disease models

The derivation of embryonic stem cells (hESC) from human embryos a decade ago started a new era in perspectives for cell therapy as well as understanding human development and disease. More recently, reprogramming of somatic cells to an embryonic stem cell‐like state (induced pluripotent stem cells, iPS) presented a new milestone in this area, making it possible to derive all cells types from any patients bearing specific genetic mutations. With the development of efficient differentiation protocols we are now able to use the derivatives of pluripotent stem cells to study mechanisms of disease and as human models for drug and toxicology testing. In addition derivatives of pluripotent stem cells are now close to be used in clinical practice although for the heart, specific additional challenges have been identified that preclude short‐term application in cell therapy. Here we review techniques presently used to induce differentiation of pluripotent stem cells into cardiomyocytes and the potential these cells have as disease models and for therapy. J. Cell. Biochem. 107: 592–599, 2009. © 2009 Wiley‐Liss, Inc.     

Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral‐nerve injury: An advantageous alternative to ES and iPS cells

The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral‐, plasmid‐ or transposon‐mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K‐15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary‐acidic‐protein (GFAP)‐positive Schwann cells and promoted the recovery of pre‐existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells. J. Cell. Biochem. 107: 1016–1020, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of single chain antibodies to breast cancer stem cells using phage display

Recent evidence suggests that most malignancies are driven by “cancer stem cells” sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. Furthermore these cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. These studies theorize that therapies, which effectively treat the majority of tumor cells but ‘miss’ the stem cell population, will fail, while therapies directed at stern cells can potentially eradicate tumors. In breast cancer, researchers have isolated ‘breast cancer stem cells’ capable of recreating the tumor in vivo and in vitro. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self‐renewal and differentiation characteristics. In the present study, an antibody phage library has been used to search for phage displayed‐single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. We demonstrate evidence of two clones binding specifically to a cancer stem cell population isolated from the SUMl59 breast cancer cell line. These clones had selective affinity for cancer stem cells and they were able to select cancer stem cells among a large population of non‐stem cancer cells in paraffin‐embedded sections. The applicability of these clones to paraffin sections and frozen tissue specimens made them good candidates to be used as diagnostic and prognostic markers in breast cancer patient samples taking into consideration the cancer stern cell concept in tumor biology. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The relative potency and safety of endothelial progenitor cells and unselected mononuclear cells for recovery from myocardial infarction and ischemia

Endothelial progenitor cells (EPCs) are a subset of the total mononuclear cell population (tMNCs) that possess an enhanced potential for differentiation within the endothelial‐cell lineage. Typically, EPCs are selected from tMNCs via the expression of both hematopoietic stem‐cell markers and endothelial‐cell markers, such as CD34, or by culturing tMNCs in media selective for endothelial cells. Both EPCs and tMNCs participate in vascular growth and regeneration, and their potential use for treatment of myocardial injury or disease has been evaluated in early‐phase clinical studies. Direct comparisons between EPCs and tMNCs are rare, but the available evidence appears to favor EPCs, particularly CD34+ cells, and the potency of EPCs may be increased as much as 30‐fold through genetic modification. However, these observations must be interpreted with caution because clinical investigations of EPC therapy are ongoing. We anticipate that with continued development, EPC therapy will become a safe and effective treatment option for patients with acute myocardial infarction or chronic ischemic disease. J. Cell. Physiol. 219: 235–242, 2009. © 2008 Wiley‐Liss, Inc.     

The clinical potential of stem cells

Stem cells are defined by their capacity for self-renewal and multilineage differentiation, making them uniquely situated to treat a broad spectrum of human diseases. For example, because hematopoietic stem cells can reconstitute the entire blood system, bone marrow transplantation has long been used in the clinic to treat various diseases. Similarly, the transplantation of other tissue-specific stem cells, such as stem cells isolated from epithelial and neural tissues, can treat mouse disease models and human patients in which epithelial and neural cells are damaged. An alternative to tissue-specific stem cell therapy takes advantage of embryonic stem cells, which are capable of differentiating into any tissue type. Furthermore, nuclear transfer, the transfer of a post-mitotic somatic cell nucleus into an enucleated oocyte, creates a limitless source of autologous cells that, when combined with gene therapy, can serve as a powerful therapeutic tool.     

Pancreatic cancer stem cells and relevance to cancer treatments

Pancreatic cancer continues to be a malignancy with few therapeutic options. The majority of patients that present for an evaluation have locally advanced or metastatic disease that is incurable by surgical approaches. Chemotherapy and radiotherapy resistance of pancreatic adenocarcinomas limits the efficacy of these therapeutic approaches. Recent evidence supports the existence of human pancreatic cancer stem cells, which appear to drive tumor initiation and progression and are particularly resistant to cell death induced by radiation or chemotherapy. Understanding the mechanisms of pancreatic cancer stem cell self‐renewal and resistance to standard therapies may lead to new, more effective therapies to treat this dismal disease. J. Cell. Biochem. 107: 40–45, 2009. © 2009 Wiley‐Liss, Inc.     

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells

Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope‐coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell‐based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 ( http://proteomecentral.proteomexchange.org/dataset/PXD001768 ).     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

Identification and expansion of human osteosarcoma‐cancer‐stem cells by long‐term 3‐aminobenzamide treatment

A novel cancer stem‐like cell line (3AB‐OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG‐63 cells by long‐term treatment (100 days) with 3‐aminobenzamide (3AB). 3AB‐OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle‐shaped, polygonal‐shaped, and rounded‐shaped. With respect to MG‐63 cells, 3AB‐OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self‐renewal ability and much higher levels of cell cycle markers which account for G1‐S/G2‐M phases progression. Differently from MG‐63 cells, 3AB‐OS cells can be reseeded unlimitedly without losing their proliferative potential. They show an ATP‐binding cassette transporter ABCG2‐dependent phenotype with high drug efflux capacity, and a strong positivity for CD133, marker for pluripotent stem cells, which are almost unmeasurable in MG‐63 cells. 3AB‐OS cells are much less committed to osteogenic and adipogenic differentiation than MG‐63 cells and highly express genes required for maintaining stem cell state (Oct3/4, hTERT, nucleostemin, Nanog) and for inhibiting apoptosis (HIF‐1α, FLIP‐L, Bcl‐2, XIAP, IAPs, and survivin). 3AB‐OS may be a novel tumor cell line useful for investigating the mechanisms by which stem cells enrichment may be induced in a tumor cell line. The identification of a subpopulation of cancer stem cells that drives tumorigenesis and chemoresistance in osteosarcoma may lead to prognosis and optimal therapy determination. Expression patterns of stem cell markers, especially CD133 and ABCG2, may indicate the undifferentiated state of osteosarcoma tumors, and may correlate with unfavorable prognosis in the clinical setting. J. Cell. Physiol. 219: 301–313, 2009. © 2009 Wiley‐Liss, Inc.     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

Human ES and iPS cells as cell sources for the treatment of Parkinson's disease: Current state and problems

Cell therapy using human embryonic stem cells (hESCs) is a promising therapeutic option for Parkinson's disease (PD), an incurable neurodegenerative disease. A prerequisite for clinical application of hESCs for PD is an efficient and strict differentiation of hESCs into midbrain dopamine (mDA) neuron‐like cells, which would be directly translated into high effectiveness of the therapy with minimum risk of undesirable side effects. Due to fruitful efforts from many laboratories, a variety of strategies for improving efficiency of dopaminergic differentiation from hESCs have been developed, mostly by optimizing culture conditions, genetic modification, and modulating intracellular signaling pathways. The rapid advances in the fields of dopaminergic differentiation of hESCs, prevention of tumor formation, and establishment of safe human induced pluripotent stem cells (hiPSCs) would open the door to highly effective, tumor‐free, and immune rejection‐free cell therapy for PD in the near future. J. Cell. Biochem. 109: 292–301, 2010. © 2009 Wiley‐Liss, Inc.     

Bioassembly of three‐dimensional embryonic stem cell‐scaffold complexes using compressed gases

Tissues are composed of multiple cell types in a well‐organized three‐dimensional (3D) microenvironment. To faithfully mimic the tissue in vivo, tissue‐engineered constructs should have well‐defined 3D chemical and spatial control over cell behavior to recapitulate developmental processes in tissue‐ and organ‐specific differentiation and morphogenesis. It is a challenge to build a 3D complex from two‐dimensional (2D) patterned structures with the presence of cells. In this study, embryonic stem (ES) cells grown on polymeric scaffolds with well‐defined microstructure were constructed into a multilayer cell‐scaffold complex using low pressure carbon dioxide (CO₂) and nitrogen (N₂). The mouse ES cells in the assembled constructs were viable, retained the ES cell‐specific gene expression of Oct‐4, and maintained the formation of embryoid bodies (EBs). In particular, cell viability was increased from 80% to 90% when CO₂ was replaced with N₂. The compressed gas‐assisted bioassembly of stem cell‐polymer constructs opens up a new avenue for tissue engineering and cell therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Muscle-derived stem cells: potential for muscle regeneration

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disease characterized by progressive muscle weakness caused by the lack of dystrophin expression at the sarcolemma of muscle fibers. Although various approaches to delivering dystrophin in dystrophic muscle have been investigated extensively (e.g., cell and gene therapy), there is still no treatment that alleviates the muscle weakness in this common inherited muscle disease. The transplantation of myoblasts can enable transient delivery of dystrophin and improve the strength of injected dystrophic muscle, but this approach has various limitations, including immune rejection, poor cellular survival rates, and the limited spread of the injected cells. The isolation of muscle cells that can overcome these limitations would enhance the success of myoblast transplantation significantly. The efficiency of cell transplantation might be improved through the use of stem cells, which display unique features, including (1) self-renewal with production of progeny, (2) appearance early in development and persistence throughout life, and (3) long-term proliferation and multipotency. For these reasons, the development of muscle stem cells for use in transplantation or gene transfer (ex vivo approach) as treatment for patients with muscle disorders has become more attractive in the past few years. In this paper, we review the current knowledge regarding the isolation and characterization of stem cells isolated from skeletal muscle by highlighting their biological features and their relationship to satellite cells as well as other populations of stem cells derived from other tissues. We also describe the remarkable ability of stem cells to regenerate skeletal muscle and their potential use to alleviate the muscle weakness associated with DMD.