FOS licenses early events in stem cell activation driving skeletal muscle regeneration.

Muscle satellite cells (SCs) are a quiescent (non-proliferative) stem cell population in uninjured skeletal muscle. Although SCs have been investigated for nearly 60 years, the molecular drivers that transform quiescent SCs into the rapidly dividing (activated) stem/progenitor cells that mediate muscle repair after injury remain largely unknown. Here we identify a prominent FBJ osteosarcoma oncogene (Fos) mRNA and protein signature in recently activated SCs that is rapidly, heterogeneously, and transiently induced by muscle damage. We further reveal a requirement for FOS to efficiently initiate key stem cell functions, including cell cycle entry, proliferative expansion, and muscle regeneration, via induction of "pro-regenerative" target genes that stimulate cell migration, division, and differentiation. Disruption of one of these Fos/AP-1 targets, NAD(+)-consuming mono-ADP-ribosyl-transferase 1 (Art1), in SCs delays cell cycle entry and impedes progenitor cell expansion and muscle regeneration. This work uncovers an early-activated FOS/ART1/mono-ADP-ribosylation (MARylation) pathway that is essential for stem cell-regenerative responses.

Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells.

Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential.

Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.

Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months, even without astrocyte feeder layers.

Inhibition of JAK-STAT signaling stimulates adult satellite cell function.

Diminished regenerative capacity of skeletal muscle occurs during adulthood. We identified a reduction in the intrinsic capacity of mouse adult satellite cells to contribute to muscle regeneration and repopulation of the niche. Gene expression analysis identified higher expression of JAK-STAT signaling targets in 3-week [corrected] 18-month-old mice [corrected]. Knockdown of Jak2 or Stat3 significantly stimulated symmetric satellite stem cell divisions on cultured myofibers. Genetic knockdown of Jak2 or Stat3 expression in prospectively isolated satellite cells markedly enhanced their ability to repopulate the satellite cell niche after transplantation into regenerating tibialis anterior muscle. Pharmacological inhibition of Jak2 and Stat3 activity similarly stimulated symmetric expansion of satellite cells in vitro and their engraftment in vivo. Intramuscular injection of these drugs resulted in a marked enhancement of muscle repair and force generation after cardiotoxin injury. Together these results reveal age-related intrinsic properties that functionally distinguish satellite cells and suggest a promising therapeutic avenue for the treatment of muscle-wasting diseases.

Canonical Wnt signaling induces a primitive endoderm metastable state in mouse embryonic stem cells.

Activation of the canonical Wnt signaling pathway synergizes with leukemia inhibitory factor (LIF) to maintain pluripotency of mouse embryonic stem cells (mESCs). However, in the absence of LIF, Wnt signaling is unable to maintain ESCs in the undifferentiated state. To investigate the role of canonical Wnt signaling in pluripotency and lineage specification, we expressed Wnt3a in mESCs and characterized them in growth and differentiation. We found that activated canonical Wnt signaling induced the formation of a reversible metastable primitive endoderm state in mESC. Upon subsequent differentiation, Wnt3a-stimulated mESCs gave rise to large quantities of visceral endoderm. Furthermore, we determined that the ability of canonical Wnt signaling to induce a metastable primitive endoderm state was mediated by Tbx3. Our data demonstrates a specific role for canonical Wnt signaling in promoting pluripotency while at the same time priming cells for subsequent differentiation into the primitive endoderm lineage.