Stem cell secretome treatment improves whole-body metabolism, reduces adiposity, and promotes skeletal muscle function in aged mice.

Aging coincides with the progressive loss of muscle mass and strength, increased adiposity, and diminished physical function. Accordingly, interventions aimed at improving muscle, metabolic, and/or physical health are of interest to mitigate the adverse effects of aging. In this study, we tested a stem cell secretome product, which contains extracellular vesicles and growth, cytoskeletal remodeling, and immunomodulatory factors. We examined the effects of 4 weeks of 2×/week unilateral intramuscular secretome injections (quadriceps) in ambulatory aged male C57BL/6 mice (22-24 months) compared to saline-injected aged-matched controls. Secretome delivery substantially increased whole-body lean mass and decreased fat mass, corresponding to higher myofiber cross-sectional area and smaller adipocyte size, respectively. Secretome-treated mice also had greater whole-body physical function (grip strength and rotarod performance) and had higher energy expenditure and physical activity levels compared to control mice. Furthermore, secretome-treated mice had greater skeletal muscle Pax7+ cell abundance, capillary density, collagen IV turnover, reduced intramuscular lipids, and greater Akt and hormone sensitive lipase phosphorylation in adipose tissue. Finally, secretome treatment in vitro directly enhanced muscle cell growth and IL-6 production, and in adipocytes, it reduced lipid content and improved insulin sensitivity. Moreover, indirect treatment with secretome-treated myotube culture media also enhanced muscle cell growth and adipocyte size reduction. Together, these data suggest that intramuscular treatment with a stem cell secretome improves whole-body metabolism, physical function, and remodels skeletal muscle and adipose tissue in aged mice.

Graft conditioning with fluticasone propionate reduces graft-versus-host disease upon allogeneic hematopoietic cell transplantation in mice.

Hematopoietic cell transplantation (HCT) treats many blood conditions but remains underused due to complications such as graft-versus-host disease (GvHD). In GvHD, donor immune cells attack the patient, requiring powerful immunosuppressive drugs like glucocorticoids (GCs) to prevent death. In this study, we tested the hypothesis that donor cell conditioning with the glucocorticoid fluticasone propionate (FLU) prior to transplantation could increase hematopoietic stem cell (HSC) engraftment and reduce GvHD. Murine HSCs treated with FLU had increased HSC engraftment and reduced severity and incidence of GvHD after transplantation into allogeneic hosts. While most T cells died upon FLU treatment, donor T cells repopulated in the hosts and appeared less inflammatory and alloreactive. Regulatory T cells (Tregs) are immunomodulatory and survived FLU treatment, resulting in an increased ratio of Tregs to conventional T cells. Our results implicate an important role for Tregs in maintaining allogeneic tolerance in FLU-treated grafts and suggest a therapeutic strategy of pre-treating donor cells (and not the patients directly) with GCs to simultaneously enhance engraftment and reduce GvHD upon allogeneic HCT.

Presentation of Human Neural Stem Cell Antigens Drives Regulatory T Cell Induction.

Transplantation of human neural stem cells (hNSCs) is a promising regenerative therapy to promote remyelination in patients with multiple sclerosis (MS). Transplantation of hNSCs has been shown to increase the number of CD4+CD25+Foxp3+ T regulatory cells (Tregs) in the spinal cords of murine models of MS, which is correlated with a strong localized remyelination response. However, the mechanisms by which hNSC transplantation leads to an increase in Tregs in the CNS remains unclear. We report that hNSCs drive the conversion of T conventional (Tconv) cells into Tregs in vitro. Conversion of Tconv cells is Ag driven and fails to occur in the absence of TCR stimulation by cognate antigenic self-peptides. Furthermore, CNS Ags are sufficient to drive this conversion in the absence of hNSCs in vitro and in vivo. Importantly, only Ags presented in the thymus during T cell selection drive this Treg response. In this study, we investigate the mechanisms by which hNSC Ags drive the conversion of Tconv cells into Tregs and may provide key insight needed for the development of MS therapies.

Regulatory T cells promote remyelination in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis following human neural stem cell transplant.

Multiple sclerosis (MS) is a chronic, inflammatory autoimmune disease that affects the central nervous system (CNS) for which there is no cure. In MS, encephalitogenic T cells infiltrate the CNS causing demyelination and neuroinflammation; however, little is known about the role of regulatory T cells (Tregs) in CNS tissue repair. Transplantation of neural stem and progenitor cells (NSCs and NPCs) is a promising therapeutic strategy to promote repair through cell replacement, although recent findings suggest transplanted NSCs also instruct endogenous repair mechanisms. We have recently described that dampened neuroinflammation and increased remyelination is correlated with emergence of Tregs following human NPC transplantation in a murine viral model of immune-mediated demyelination. In the current study we utilized the prototypic murine autoimmune model of demyelination experimental autoimmune encephalomyelitis (EAE) to test the efficacy of hNSC transplantation. Eight-week-old, male EAE mice receiving an intraspinal transplant of hNSCs during the chronic phase of disease displayed remyelination, dampened neuroinflammation, and an increase in CNS CD4+CD25+FoxP3+ regulatory T cells (Tregs). Importantly, ablation of Tregs abrogated histopathological improvement. Tregs are essential for maintenance of T cell homeostasis and prevention of autoimmunity, and an emerging role for Tregs in maintenance of tissue homeostasis through interactions with stem and progenitor cells has recently been suggested. The data presented here provide direct evidence for collaboration between CNS Tregs and hNSCs promoting remyelination.

Developing a clinical-grade cryopreservation protocol for human testicular tissue and cells.

Recent work in preservation of female fertility as well as new information on the nature of spermatogonial stem cells has prompted an investigation into the possibility of an effective clinical-grade procedure for the cryopreservation of testicular cells and/or tissue. Clinical-grade reagents, validated equipment, and protocols consistent with cGTP/cGMP standards were used in developing a procedure suitable for the safe and effective cryopreservation of human testicular cells and tissues. These procedures were designed to be compliant with the relevant FDA regulations. The procedure proved to effectively cryopreserve both testicular cells and tissue. The cryopreservation of testicular tissue was comparable in most aspects we measured to the cryopreservation of isolated cells, except that the viability of the cells from cryopreserved testicular tissue was found to be significantly higher. On the other hand, cryopreservation of cells is preferred for cell analysis, quality control, and sterility testing. This study demonstrates that testicular tissue and cells from sexual reassignment patients can be successfully cryopreserved with a clinical-grade procedure and important cell populations are not only preserved but also enriched by the process. Further studies will determine whether these findings from hormone-treated patients can be generalized to other patients.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis.

BACKGROUND: This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. METHODS: Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. RESULTS: Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. CONCLUSIONS: This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.