Barriers in contribution of human mesenchymal stem cells to murine muscle regeneration.

AIM: To study regeneration of damaged human and murine muscle implants and the contribution of added xenogeneic mesenchymal stem cells (MSCs). METHODS: Minced human or mouse skeletal muscle tissues were implanted together with human or mouse MSCs subcutaneously on the back of non-obese diabetic/severe combined immunodeficient mice. The muscle tissues (both human and murine) were minced with scalpels into small pieces (< 1 mm(3)) and aliquoted in portions of 200 mm(3). These portions were either cryopreserved in 10% dimethylsulfoxide or freshly implanted. Syngeneic or xenogeneic MSCs were added to the minced muscles directly before implantation. Implants were collected at 7, 14, 30 or 45 d after transplantation and processed for (immuno)histological analysis. The progression of muscle regeneration was assessed using a standard histological staining (hematoxylin-phloxin-saffron). Antibodies recognizing Pax7 and von Willebrand factor were used to detect the presence of satellite cells and blood vessels, respectively. To enable detection of the bone marrow-derived MSCs or their derivatives we used MSCs previously transduced with lentiviral vectors expressing a cytoplasmic LacZ gene. X-gal staining of the fixed tissues was used to detect ß-galactosidase-positive cells and myofibers. RESULTS: Myoregeneration in implants of fresh murine muscle was evident as early as day 7, and progressed with time to occupy 50% to 70% of the implants. Regeneration of fresh human muscle was slower. These observations of fresh muscle implants were in contrast to the regeneration of cryopreserved murine muscle that proceeded similarly to that of fresh tissue except for day 45 (P < 0.05). Cryopreserved human muscle showed minimal regeneration, suggesting that the freezing procedure was detrimental to human satellite cells. In fresh and cryopreserved mouse muscle supplemented with LacZ-tagged mouse MSCs, ß-galactosidase-positive myofibers were identified early after grafting at the well-vascularized periphery of the implants. The contribution of human MSCs to murine myofiber formation was, however, restricted to the cryopreserved mouse muscle implants. This suggests that fresh murine muscle tissue provides a suboptimal environment for maintenance of human MSCs. A detailed analysis of the histological sections of the various muscle implants revealed the presence of cellular structures with a deviating morphology. Additional stainings with alizarin red and alcian blue showed myofiber calcification in 50 of 66 human muscle implants, and encapsulated cartilage in 10 of 81 of murine muscle implants, respectively. CONCLUSION: In mouse models the engagement of human MSCs in myoregeneration might be underestimated. Furthermore, our model permits the dissection of species-specific factors in the microenvironment.

Prospects and challenges of induced pluripotent stem cells as a source of hematopoietic stem cells.

Many life-threatening hematological diseases are now treated by bone marrow transplantations, i.e., infusion of hematopoietic stem cells (HSCs). HSC transplantations are a valid option for the treatment of a variety of metabolic disorders, and even for solid tumors and some refractory severe autoimmune diseases. Unfortunately, the frequency and outcome of HSC transplantations are limited by a shortage of suitable donors. Induced pluripotent stem cells (iPSCs)--somatic cells that have acquired pluripotent stem cell characteristics by the ectopic expression of pluripotency-inducing factors--have been proposed as an alternative source of HSCs. Possible applications include cells of autologous, of autologous and genetically modified, or of allogeneic origin. Here, we provide a perspective on the distinct opportunities of iPSCs and discuss the challenges that lie ahead.

Myogenic properties of human mesenchymal stem cells derived from three different sources.

Mesenchymal stem cells (MSCs) of mammals have been isolated from many tissues and are characterized by their aptitude to differentiate into bone, cartilage, and fat. Differentiation into cells of other lineages like skeletal muscle, tendon/ligament, nervous tissue, and epithelium has been attained with MSCs derived from some tissues. Whether such abilities are shared by MSCs of all tissues is unknown. We therefore compared for three human donors the myogenic properties of MSCs from adipose tissue (AT), bone marrow (BM), and synovial membrane (SM). Our data show that human MSCs derived from the three tissues differ in phenotype, proliferation capacity, and differentiation potential. The division rate of AT-derived MSCs (AT-MSCs) was distinctly higher than that of MSCs from the other two tissue sources. In addition, clear donor-specific differences in the long-term maintenance of MSC proliferation ability were observed. Although similar in their in vitro fusogenic capacity with murine myoblasts, MSCs of the three sources contributed to a different extent to skeletal muscle regeneration in vivo. Transplanting human AT-, BM-, or SM-MSCs previously transduced with a lentiviral vector encoding ß-galactosidase into cardiotoxin-damaged tibialis anterior muscles (TAMs) of immunodeficient mice revealed that at 30 days after treatment the frequency of hybrid myofibers was highest in the TAMs treated with AT-MSCs. Our finding of human-specific ß-spectrin and dystrophin in hybrid myofibers containing human nuclei argues for myogenic programming of MSCs in regenerating murine skeletal muscle. For the further development of MSC-based treatments of myopathies, AT-MSCs appear to be the best choice in view of their efficient contribution to myoregeneration, their high ex vivo expansion potential, and because their harvesting is less demanding than that of BM- or SM-MSCs.

Pressure ulcers: description of a new model and use of mesenchymal stem cells for repair.

BACKGROUND: Pressure ulcers (PUs) still represent a heavy burden on many patients and nursing institutions. Our understanding of the pathophysiology and development of new treatments are hampered by the scarcity of suitable animal models. OBJECTIVE: Evaluation of the translational value of an easily accessible mouse model. METHODS: PUs were induced by application of magnetic devices on the dorsal skin of mice, which causes localized ischemia. The extent of the lesions and healing rate were quantified. Variations in ischemic exposure time were compared in hairless and normal mice. A detailed histological analysis of regeneration is presented. The influence of streptozotocin-induced diabetes, skin X-irradiation and treatment of the ulcers with human mesenchymal stem cells (MSCs) was investigated using immunodeficient NOD/SCID mice. RESULTS: Ulcers induced by this form of ischemia have many features in common with decubitus ulcers in humans. No difference between hairy and hairless mice was observed in the rate of healing of the PUs. Unexpectedly, healing was not delayed in diabetic mice, but skin X-irradiation prior to ischemia resulted in a doubling of the time to complete closure of the PUs, and delayed repair of the dermis and panniculus carnosus muscle. Intradermal transplantation of human MSCs did not accelerate healing. The grafted MSCs were short-lived and only marginally participated in regeneration by differentiating into tissue-specific cells. CONCLUSION: The results emphasize the difference in the characteristics of PUs as compared to surgical wounds. This experimental model is recommended for preclinical research on decubitus ulcers because of its mechanistic similarity with clinical PUs and its simplicity.

Exploitation of herpesvirus immune evasion strategies to modify the immunogenicity of human mesenchymal stem cell transplants.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected. METHODOLOGY/PRINCIPAL FINDINGS: We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable.

Long-term contribution of human bone marrow mesenchymal stromal cells to skeletal muscle regeneration in mice.

Mesenchymal stromal cells (MSCs) are attractive for cellular therapy of muscular dystrophies as they are easy to procure, can be greatly expanded ex vivo, and contribute to skeletal muscle repair in vivo. However, detailed information about the contribution of bone marrow (BM)-derived human MSCs (BM-hMSCs) to skeletal muscle regeneration in vivo is very limited. Here, we present the results of a comprehensive study of the fate of LacZ-tagged BM-hMSCs following implantation in cardiotoxin (CTX)-injured tibialis anterior muscles (TAMs) of immunodeficient mice. ß-Galactosidase-positive (ß-gal(+)) human-mouse hybrid myofibers (HMs) were counted in serial cross sections over the full length of the treated TAMs of groups of mice at monthly intervals. The number of human cells was estimated using chemiluminescence assays. While the number of human cells declined gradually to about 10% of the injected cells at 60 days after transplantation, the number of HMs increased from day 10 onwards, reaching 104 ± 39.1 per TAM at 4 months postinjection. ß-gal(+) cells and HMs were distributed over the entire muscle, indicating migration of the former from the central injection site to the ends of the TAMs. The identification of HMs that stained positive for human spectrin suggests myogenic reprogramming of hMSC nuclei. In summary, our findings reveal that BM-hMSCs continue to participate in the regeneration/remodeling of CTX-injured TAMs, resulting in ±5% HMs at 4 months after damage induction. Moreover, donor-derived cells were shown to express genetic information, both endogenous and transgenic, in recipient myofibers.

Anomer-equilibrated streptozotocin solution for the induction of experimental diabetes in mice (Mus musculus).

Streptozotocin is widely used to induce diabetes in laboratory animals through multiple low-dose or single high-dose intraperitoneal injections. HPLC analysis has shown that the composition of the solution may change considerably during the first 2 h after dissolution due to equilibration of the 2 anomers (alpha and beta) of streptozotocin. Because of the drug's alleged instability in solution, the typical recommendation is to administer streptozotocin within 10 min after dissolution. We compared the induction of diabetes in NOD/SCID mice by injection of a single high dose of freshly made or anomer-equilibrated streptozotocin solution. Solutions were prepared from dry compound containing 85% of the alpha anomer, which is the more toxic of the 2. Body weight and nonfasting blood glucose levels were measured weekly for 8 wk. Both solutions induced long-term hyperglycemia, but blood glucose levels and mortality were higher and damage to pancreatic islands more pronounced in the mice receiving freshly prepared solution. A small proportion of mice did not respond in both treatment groups. If stored at 4 degrees C in the dark, the anomer-equilibrated solution retains its biologic activity for at least 40 d; under those conditions the streptozotocin content decreases by 0.1% daily, as determined by HPLC. Anomer-equilibrated streptozotocin solution has several practical advantages, and we recommend its use as standard for the induction of experimental diabetes because this practice may improve reproducibility and comparison of results between different laboratories.

Phenotypic and functional reversal within the early human hematopoietic compartment.

The fate of phenotypically defined human hematopoietic stem cells (hHSCs) in culture and the link between their surface marker expression profile and function are still controversial. We studied these aspects of hHSC biology by relating the expression of the early lineage markers (ELM) CD33, CD38, and CD71 on the surface of human umbilical cord blood (UCB) CD34(+) cells to their long-term nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse repopulation activity (LT-SRA). In uncultured UCB samples, LT-SRA was largely confined to the small CD34(+)ELM(-) cell fraction. CD34(+) cells expressing ELM markers at their surface usually lacked LT-SRA. After culturing UCB CD34(+) cells for 6 days in serum-free medium and on a feeder layer of Rat2 cells, the number of CD34(+)ELM(-) cells stayed roughly the same or showed a slight increase and the LT-SRA was preserved, suggesting a close association between LT-SRA and the CD34(+)ELM(-) phenotype. Indeed, transplantation of CD34(+)ELM(-) cells isolated from cultured UCB CD34(+) cells resulted in long-term hematopoietic reconstitution of conditioned NOD/SCID mice, whereas CD34(+)ELM(+) cells derived from the same cultures were devoid of LT-SRA. Remarkably, roughly 1% of the cells recovered from cultures initiated with isolated CD34(+)ELM(+) cells had lost ELM surface expression. Concurrently, the cultured CD34(+)ELM(+) cells acquired LT-SRA, suggesting that hematopoietic stem cells (HSCs) may arise by the dedifferentiation of early hematopoietic progenitor cells. The latter finding challenges the paradigm of unidirectional hematopoietic differentiation and opens new opportunities for HSC expansion prior to transplantation.

Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific integration ability.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene, making it a potential target for gene therapy. There is, however, a scarcity of vectors that can accommodate the 14-kb DMD cDNA and permanently genetically correct muscle tissue in vivo or proliferating myogenic progenitors in vitro for use in autologous transplantation. Here, a dual high-capacity adenovirus-adeno-associated virus (hcAd/AAV) vector with two full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements is presented. These vectors are generated from input linear monomeric DNA molecules consisting of the Ad origin of replication and packaging signal followed by the recently identified AAV DNA integration efficiency element (p5IEE), the transgene(s) of interest, and the AAV inverted terminal repeat (ITR). After infection of producer cells with a helper Ad vector, the Ad DNA replication machinery, in concert with the AAV ITR-dependent dimerization, leads to the assembly of vector genomes with a tail-to-tail configuration that are efficiently amplified and packaged into Ad capsids. These dual hcAd/AAV hybrid vectors were used to express the dystrophin-coding sequence in rat cardiomyocytes in vitro and to restore dystrophin synthesis in the muscle tissues of mdx mice in vivo. Introduction into human cells of chimeric genomes, which contain a structure reminiscent of AAV proviral DNA, resulted in AAV Rep-dependent targeted DNA integration into the AAVS1 locus on chromosome 19. Dual hcAd/AAV hybrid vectors may thus be particularly useful to develop safe treatment modalities for diseases such as DMD that rely on efficient transfer and stable expression of large genes.

Stem cell transplantation for autoimmune disorders. Preclinical experiments.

The clinical use of autologous stem cell transplants for the treatment of refractory severe autoimmune diseases was preceded by convincing proof of its underlying principle in animal models. The various categories of experimental autoimmune disease in laboratory rodents are briefly described here, and the rationale that was used in the selection of suitable experimental autoimmune diseases for translational research is explained. The two models that provided the bulk of the data needed for designing the initial clinical treatment protocols were adjuvant arthritis (AA) and experimental allergic encephalomyelitis (EAE), which were both induced in Buffalo rats. In this strain, AA is manifested as a chronic, progressive, systemic polyarthritis and EAE as a chronic, remitting/relapsing form of encephalomyelitis resembling multiple sclerosis. Both diseases can be cured with autologous stem cell transplantation provided that adequate conditioning is given and that the disease has not yet progressed to the stage of 'scarring'. It is basically the inflammatory stages that respond well to this therapy. The success of treatment depends on how completely the autoantigen-specific activated T-lymphocytes and memory cells are eradicated. Because of a lack of information on the nature of the autoantigens involved in human disease and on the size of those cell populations in the animal models as well as in humans, this aspect of translation is difficult. The experiments have, however, provided important guidelines. High-dose conditioning regimens yield better results than low-dose conditioning, certain conditioning agents perform better than others, and care should be taken not to reintroduce too many T-cells with the autologous graft. The clinical results obtained so far indicate a high predictive power of these two animal models, which are therefore recommended strongly for additional preclinical studies.

Phylogenetic aspects of tissue regeneration: role of stem cells. A concise overview.

Many different forms of regeneration are known among the members of the animal kingdom. Invertebrates commonly generate new individuals by sprouting and splitting off of body parts, so that the processes of asexual reproduction and of regeneration as a response to injury can hardly be distinguished. Among the adult vertebrates, regeneration of lost body parts has become exceptional rather than the rule. However, the capacity for regeneration of tissues and for the remodeling of injured organs is much better preserved than is generally appreciated. The main reason for this misconception is the enormous variety of mechanisms for replenishment, repair, and remodeling that coexist in one and the same animal. In addition, there is a considerable variation in the response to damage inflicted by different forms of injury. Our conceptions of wound repair and tissue regeneration have been radically changed by the recent discoveries of stem cell plasticity and of dedifferentiation of supposed end cells in mice and men. Seemingly, these mechanisms should provide our bodies with near inexhaustible powers of regeneration. Yet, failures to achieve just that are among the most pressing problems of modern medicine. Apparently, counterforces have evolved that prevent the orderly replacement of lost cells. The present challenge for regenerative medicine is to unravel those barriers and find means to overcome them.