Geometric control of myogenic cell fate.

This work combines expertise in stem cell biology and bioengineering to define the system for geometric control of proliferation and differentiation of myogenic progenitor cells. We have created an artificial niche of myogenic progenitor cells, namely, modified extracellular matrix (ECM) substrates with spatially embedded growth or differentiation factors (GF, DF) that predictably direct muscle cell fate in a geometric pattern. Embedded GF and DF signal progenitor cells from specifically defined areas on the ECM successfully competed against culture media for myogenic cell fate determination at a clearly defined boundary. Differentiation of myoblasts into myotubes is induced in growth-promoting medium, myotube formation is delayed in differentiation-promoting medium, and myogenic cells, at different stages of proliferation and differentiation, can be induced to coexist adjacently in identical culture media. This method can be used to identify molecular interactions between cells in different stages of myogenic differentiation, which are likely to be important determinants of tissue repair. The designed ECM niches can be further developed into a vehicle for transplantation of myogenic progenitor cells maintaining their regenerative potential. Additionally, this work may also serve as a general model to engineer synthetic cellular niches to harness the regenerative potential of organ stem cells

Comparative Angiogenic Activities of Induced Pluripotent Stem Cells Derived from Young and Old Mice

Advanced age is associated with decreased stem cell activity. However, the effect of aging on the differentiation capacity of induced pluripotent stem (iPS) cells into cardiovascular cells has not been fully clarified. We investigated whether iPS cells derived from young and old mice are equally capable of differentiating into vascular progenitor cells, and whether these cells regulate vascular responses in vivo. iPS cells from mouse embryonic fibroblasts (young) or 21 month-old mouse bone marrow (old) were used. Fetal liver kinase-1 positive (Flk-1+) cells, as a vascular progenitor marker, were induced after 3 to 4 days of culture from iPS cells derived from young and old mice. These Flk-1+ cells were sorted and shown to differentiate into VE-cadherin+ endothelial cells and α-SMA+ smooth muscle cells. Tube-like formation was also successfully induced in both young and old murine Flk-1+ cells. Next, hindlimb ischemia was surgically induced, and purified Flk-1+ cells were directly injected into ischemic hindlimbs of nude mice. Revascularization of the ischemic hindlimb was significantly accelerated in mice transplanted with Flk-1+ cells derived from iPS cells from either young or old mice, as compared to control mice as evaluated by laser Doppler blood flowmetry. The degree of revascularization was similar in the two groups of ischemic mice injected with iPS cell-derived Flk-1+ cells from young or old mice. Transplantation of Flk-1+ cells from both young and old murine iPS cells also increased the expression of VEGF, HGF and IGF mRNA in ischemic tissue as compared to controls. iPS cell-derived Flk-1+ cells differentiated into vascular progenitor cells, and regulated angiogenic vascular responses both in vitro and in vivo. These properties of iPS cells derived from old mice are essentially the same as those of iPS cells from young mice, suggesting the functionality of generated iPS cells themselves to be unaffected by aging

The aged niche disrupts muscle stem cell quiescence

SUMMARY The niche is a conserved regulator of stem cell quiescence and function. During aging, stem cell function declines. To what extent and by which means age-related changes within the niche contribute to this phenomenon are unknown. We demonstrate that the aged muscle stem cell niche, the muscle fiber, expresses FGF2 under homeostatic conditions, driving a subset of satellite cells to break quiescence and lose self-renewing capacity. We show that relatively dormant aged satellite cells robustly express Sprouty1 (spry1), an inhibitor of FGF signalling. Increasing FGF signalling in aged satellite cells under homeostatic conditions by removing spry1, results in the loss of quiescence, satellite cell depletion and diminished regenerative capacity. Conversely, reducing niche-derived FGF activity through inhibition of FGFR1 signalling or overexpression of spry1 in satellite cells prevents their depletion. These experiments identify an age-dependent change in the stem cell niche that directly influences stem cell quiescence and function

Wnt/β-Catenin Signaling Induces the Aging of Mesenchymal Stem Cells through the DNA Damage Response and the p53/p21 Pathway

Recent studies have demonstrated the importance of cellular extrinsic factors in the aging of adult stem cells. However, the effects of an aged cell–extrinsic environment on mesenchymal stem cell (MSC) aging and the factors involved remain unclear. In the current study, we examine the effects of old rat serum (ORS) on the aging of MSCs, and explore the effects and mechanisms of Wnt/β-catenin signaling on MSC aging induced by ORS treatment. Senescence-associated changes in the cells are examined with SA-β-galactosidase staining and ROS staining. The proliferation ability is detected by MTT assay. The surviving and apoptotic cells are determined using AO/EB staining. The results suggest that ORS promotes MSC senescence and reduces the proliferation and survival of cells. The immunofluorescence staining shows that the expression of β-catenin increases in MSCs of old rats. To identify the effects of Wnt/β-catenin signaling on MSC aging induced with ORS, the expression of β-catenin, GSK-3β, and c-myc are detected. The results show that the Wnt/β-catenin signaling in the cells is activated after ORS treatment. Then we examine the aging, proliferation, and survival of MSCs after modulating Wnt/β-catenin signaling. The results indicate that the senescence and dysfunction of MSCs in the medium containing ORS is reversed by the Wnt/β-catenin signaling inhibitor DKK1 or by β-catenin siRNA. Moreover, the expression of γ-H2A.X, a molecular marker of DNA damage response, p16INK4a, p53, and p21 is increased in senescent MSCs induced with ORS, and is also reversed by DKK1 or by β-catenin siRNA. In summary, our study indicates the Wnt/β-catenin signaling may play a critical role in MSC aging induced by the serum of aged animals and suggests that the DNA damage response and p53/p21 pathway may be the main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling

Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice

<p>Abstract</p> <p>Background</p> <p>Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients.</p> <p>Results</p> <p>In sex-mismatched transfers, functionality of female donor leukocytes was not affected in male recipients. In contrast, male donor leukocytes were greatly affected in the female recipients. In MHC-mismatches, recipients of different MHC backgrounds, or mice of different strains, showed a greater negative impact on donor leukocytes than sex-mismatches. The negative effects of sex-mismatch and MHC-mismatch on donor leukocytes were additive. Old donor leukocytes performed worse than young donor leukocytes in all settings including in young recipients. Young recipients were not able to revive the declining function of old donor leukocytes. However, the function of young donor leukocytes declined gradually in old recipients, suggesting that an aged environment may contain factors that are deleterious to cellular functions. The irradiation of donor leukocytes prior to transfers had a profound suppressive effect on donor leukocyte functions, possibly as a result of impaired transcription. The cryopreserving of donor leukocytes in liquid nitrogen had no apparent effect on donor leukocyte functions, except for a small loss of cell number after revival from freezing.</p> <p>Conclusion</p> <p>Despite the functional suppression of donor leukocytes in sex- and MHC-mismatched recipients, as well as old recipients, there was a therapeutic time period during the initial few weeks during which donor leukocytes were functional before their eventual rejection or functional decline. The eventual rejection of donor leukocytes will likely prevent donor leukocyte engraftment which would help minimize the risk of transfusion-associated graft-versus-host disease. Therefore, using leukocytes from healthy donors with high anti-cancer activity may be a feasible therapeutic concept for treating malignant diseases.</p

The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration

Inhibition of Myostatin Signaling through Notch Activation following Acute Resistance Exercise

Myostatin is a TGFb family member and negative regulator of muscle size. Due to the complexity of the molecular pathway between myostatin mRNA/protein and changes in transcription, it has been difficult to understand whether myostatin plays a role in resistance exercise-induced skeletal muscle hypertrophy. To circumvent this problem, we determined the expression of a unique myostatin target gene, Mighty, following resistance exercise. Mighty mRNA increased by 6 h (82.9624.21%) and remained high out to 48 h (56.5619.67%) after resistance exercise. Further examination of the soleus, plantaris and tibialis anterior muscles showed that the change in Mighty mRNA at 6 h correlated with the increase in muscle size associated with this protocol (R2 = 0.9996). The increase in Mighty mRNA occurred both independent of Smad2 phosphorylation and in spite of an increase in myostatin mRNA (341.86147.14% at 3 h). The myostatin inhibitor SKI remained unchanged. However, activated Notch, another potential inhibitor of TGFb signaling, increased immediately following resistance exercise (83611.2%) and stayed elevated out to 6 h (78616.6%). Electroportion of the Notch intracellular domain into the tibialis anterior resulted in an increase in Mighty mRNA (63613.4%) that was equivalent to the canonical Notch target HES-1 (94.467.32%). These data suggest that acute resistance exercise decreases myostatin signaling through the activation of the TGFb inhibitor Notch resulting in a decrease in myostatin transcriptional activity that correlates well with muscle hypertrophy

Functional Myogenic Engraftment from Mouse iPS Cells

Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies