Stem cell therapy holds significant potential for skeletal muscle repair, with in vitro-generated human muscle reserve cells (MuRCs) emerging as a source of quiescent myogenic stem cells that can be injected to enhance muscle regeneration. However, the clinical translation of such therapies is hampered by the need for fetal bovine serum (FBS) during the in vitro generation of human MuRCs. This study aimed to determine whether fresh allogeneic human platelet-rich plasma (PRP) combined or not with hyaluronic acid (PRP-HA) could effectively replace xenogeneic FBS for the ex vivo expansion and differentiation of human primary myoblasts. Cells were cultured in media supplemented with either PRP or PRP-HA and their proliferation rate, cytotoxicity and myogenic differentiation potential were compared with those cultured in media supplemented with FBS. The results showed similar proliferation rates among human myoblasts cultured in PRP, PRP-HA or FBS supplemented media, with no cytotoxic effects. Human myoblasts cultured in PRP or PRP-HA showed reduced fusion ability upon differentiation. Nevertheless, we also observed that human MuRCs generated from PRP or PRP-HA myogenic cultures, exhibited increased Pax7 expression and delayed re-entry into the cell cycle upon reactivation, indicating a deeper quiescent state of human MuRCs. These results suggest that allogeneic human PRP effectively replaces FBS for the ex vivo expansion and differentiation of human myoblasts and favors the in vitro generation of Pax7High human MuRCs, with important implications for the advancement of stem cell-based muscle repair strategies.

Reptilian skin coloration is spectacular and diverse, yet little is known about the ontogenetic processes that govern its establishment and the molecular signaling pathways that determine it. Here, we focus on the development of the banded pattern of leopard gecko hatchlings and the transition to black spots in the adult. With our histological analyses, we show that iridophores are present in the white and yellow bands of the hatchling and they gradually perish in the adult skin. Furthermore, we demonstrate that melanophores can autonomously form spots in the absence of the other chromatophores both on the regenerated skin of the tail and on the dorsal skin of the Mack Super Snow (MSS) leopard geckos. This color morph is characterized by uniform black coloration in hatchlings and black spots in adulthood; we establish that their skin is devoid of xanthophores and iridophores at both stages. Our genetic analyses identified a 13-nucleotide deletion in the PAX7 transcription factor of MSS geckos, affecting its protein coding sequence. With our single-cell transcriptomics analysis of embryonic skin, we confirm that PAX7 is expressed in iridophores and xanthophores, suggesting that it plays a key role in the differentiation of both chromatophores. Our in situ hybridizations on whole-mount embryos document the dynamics of the skin pattern formation and how it is impacted in the PAX7 mutants. We hypothesize that the melanophores-iridophores interactions give rise to the banded pattern of the hatchlings and black spot formation is an intrinsic capacity of melanophores in the postembryonic skin.

While studies have identified characteristics of quiescent satellite cells, their isolation has been hampered by the fact that the isolation procedures result in the activation of these cells into their rapidly proliferating progeny (myoblasts). Thus, the use of myoblasts for therapeutic (regenerative medicine) or industrial applications (cellular agriculture) has been impeded by the limited proliferative and differentiative capacity of these myogenic progenitors. Here we identify a subpopulation of satellite cells isolated from mouse skeletal muscle using flow cytometry that are highly Pax7-positive, exhibit a very slow proliferation rate (7.7 ± 1.2 days/doubling), and are capable of being maintained in culture for at least three months without a change in phenotype. These cells can be activated from quiescence using a p38 inhibitor or by exposure to freeze-thaw cycles. Once activated, these cells proliferate rapidly (22.7 ± 0.2 hours/doubling), have reduced Pax7 expression (3-fold decrease in Pax7 fluorescence vs. quiescence) and differentiate into myotubes with a high efficiency. Furthermore, these cells withstand freeze-thawing readily without a significant loss of viability (83.1 ± 2.1% live). The results presented here provide researchers with a method to isolate quiescent satellite cells, allowing for more detailed examinations of the factors affecting satellite cell quiescence/activation and providing a cell source that has a unique potential in the regenerative medicine and cellular agriculture fields.

BACKGROUND: Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored.
METHODS: We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources.
RESULTS: We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC.
CONCLUSIONS: Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

Lines with few or no pigment cells have been established in fishes, and these lines are useful for bioimaging. The transparent goldfish (tra) line previously established by N-ethyl-N-nitrosourea (ENU) mutagenesis is also suitable for such experiments. However, in the case of tra, leucophores form in the adult fish, making it difficult to observe the organs inside body from outside the body. In this study, we attempted to create a knockout line of the pax7a and pax7b genes, which are thought to be involved in the formation of leucophores, to further improve the transparency of tra strain.Mutations were introduced by microinjection of the CRISPR/Cas9 mixture into single-cell embryos, mutant individuals were found in F0, and the next generation was generated to confirm the mutation patterns. As a result, multiple mutation patterns, including knockout, were obtained. The same pattern of knockout F1 with pax7a and pax7b mutations was crossed to generate a homozygous knockout in F2.In the resulting pax7b-/- (tra) fish but not in pax7a-/- (tra) fish, the number of leucophores was reduced compared to that in tra, and the transparency of the body was improved. It was suggested that pax7b plays an important role in leucophore formation in goldfish. The established transparent pax7b-/- (tra) goldfish line will be a useful model for bioimaging of the body interior.

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors\' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.

The principles of myogenesis play crucial roles in the production of cultured meat, and identifying protein stimulators associated with myogenesis holds great potential to enhance the efficiency of this process. In this study, we used surface plasmon resonance (SPR)-based screening of a natural product library to discover ligands for Pax7 and MyoD, key regulators of satellite cells (SCs), and performed cell-based assays on Hanwoo SCs (HWSCs) to identify substances that promote cell proliferation and/or differentiation. Through an SPR analysis, we found that six chemicals, including one Pax7+/MyoD- chemical, four Pax7+/MyoD+ chemicals, and one Pax7-/MyoD+ chemical, bound to Pax7 and/or MyoD proteins. Among four Pax7+/MyoD+ chemicals, parthenolide (0.5 and 1 µM) and rutin (100 and 200 µM) stimulated cell proliferation in the medium with 10% FBS similar to the medium with 20% FBS, without affecting differentiation. Adenosine, a Pax7-/MyoD+ chemical, accelerated differentiation. These chemicals could be potential additives to reduce the reliance of FBS required for HWSC proliferation and differentiation in cultured meat production.

BACKGROUND: Worldwide cleft lip with or without a cleft palate (CL/P) is the most common craniofacial birth defect. Apart from changes in facial appearance, additionally affected individuals often suffer from various associated comorbidities requiring complex multidisciplinary treatment with overall high expenses. Understanding the complete pathogenetic mechanisms of CL/P might aid in developing new preventative strategies and therapeutic approaches, help with genetic counselling, and improve quality of life. Many genes have been associated with the development of orofacial clefts; however, the majority require further research. Based on the role of PAX7, PAX9, SHH, SOX3, WNT3A, and WNT9B in orofacial development, the intention was to use chromogenic in situ hybridization to detect the six genes in postnatal CLP-affected palatine tissue and compare their distribution within the tissue samples.
RESULTS: Statistically significant differences in the distribution of PAX7, PAX9, WNT3A, and WNT9B were observed. In total, 19 pairs of moderate to very strong positive correlations were noted.
CONCLUSIONS: Changes in the cleft-affected palatine epithelium primarily seem to be associated with the PAX7 gene; however, PAX9, WNT3A, WNT9B, and SOX3 role seems to be more limited. Whilst connective tissue changes seem to depend on PAX7 only, SHH seems to participate individually and indistinctly. Numerous positive correlations reflect the complicating interactions of the pathways and their components in the orofacial cleft morphopathogenesis.