The Susceptibility and Potential Functions of the LBX1 Gene in Adolescent Idiopathic Scoliosis

Genome-wide association studies have identified many susceptibility genes for adolescent idiopathic scoliosis (AIS). However, most of the results are hard to be replicated in multi-ethnic populations. LBX1 is the most promising candidate gene in the etiology of AIS. We aimed to appraise the literature for the association of LBX1 gene polymorphisms with susceptibility and curve progression in AIS. We also reviewed the function of the LBX1 gene in muscle progenitor cell migration and neuronal determination processes. Three susceptibility loci (rs11190870, rs625039, and rs11598564) near the LBX1 gene, as well as another susceptibility locus (rs678741), related to LBX1 regulation, have been successfully verified to have robust associations with AIS in multi-ethnic populations. The LBX1 gene plays an essential role in regulating the migration and proliferation of muscle precursor cells, and it is known to play a role in neuronal determination processes, especially for the fate of somatosensory relay neurons. The LBX1 gene is the most promising candidate gene in AIS susceptibility due to its position and possible functions in muscle progenitor cell migration and neuronal determination processes. The causality between susceptibility loci related to the LBX1 gene and the pathogenesis of AIS deserves to be explored with further integrated genome-wide and epigenome-wide association studies.

Lineage tracing reveals evidence of a popliteal lymphatic muscle progenitor cell that is distinct from skeletal and vascular muscle progenitors

Loss of popliteal lymphatic vessel (PLV) contractions, which is associated with damage to lymphatic muscle cells (LMCs), is a biomarker of disease progression in mice with inflammatory arthritis. Currently, the nature of LMC progenitors has yet to be formally described. Thus, we aimed to characterize the progenitors of PLV-LMCs during murine development, towards rational therapies that target their proliferation, recruitment, and differentiation onto PLVs. Since LMCs have been described as a hybrid phenotype of striated and vascular smooth muscle cells (VSMCs), we performed lineage tracing studies in mice to further clarify this enigma by investigating LMC progenitor contribution to PLVs in neonatal mice. PLVs from Cre-tdTomato reporter mice specific for progenitors of skeletal myocytes (Pax7+ and MyoD+) and VSMCs (Prrx1+ and NG2+) were analyzed via whole mount immunofluorescent microscopy. The results showed that PLV-LMCs do not derive from skeletal muscle progenitors. Rather, PLV-LMCs originate from Pax7−/MyoD−/Prrx1+/NG2+ progenitors similar to VSMCs prior to postnatal day 10 (P10), and from a previously unknown Pax7−/MyoD−/Prrx1+/NG2− muscle progenitor pathway during development after P10. Future studies of these LMC progenitors during maintenance and repair of PLVs, along with their function in other lymphatic beds, are warranted.

Consumption of a Blueberry-Enriched Diet by Women for 6 Weeks Alters Determinants of Human Muscle Progenitor Cell Function

ABSTRACT Background Human muscle progenitor cell (hMPC) function facilitates skeletal muscle regeneration and is influenced by circulating factors. Yet it is unknown whether dietary interventions impact hMPC function. Blueberry consumption was examined due to the pro-proliferative and antioxidant effects of blueberries and blueberry-derived compounds. Objectives This study measured indicators of hMPC function in young and old cultures treated with serum collected from a blueberry-enriched diet (BED) intervention. Methods Younger (21–40 y, n = 12) and older (60–79 y, n = 10) women consumed a 6-wk BED (38 g of freeze-dried blueberries daily). Fasting serum was collected at 0, 4, and 6 wk, and a fed serum sample at 1.5 h (acute) after starting the BED intervention. Young and old hMPCs, derived from 3–5 distinct donors (biological replicates), were individually cultured in media containing pooled, age-group–matched serum from each time point. Determinants of hMPC function (e.g., hMPC number, oxidative stress resistance, and upregulation of metabolic pathways) were measured and compared within age groups. Results Culturing young hMPCs in acute (compared with 0 wk) BED serum did not alter hMPC number or oxidative stress–induced cell death, but increased cellular oxygen consumption (29%, P = 0.026). Culturing young hMPCs in 6-wk (compared with 0-wk) BED serum increased hMPC number (40%, P = 0.0024), conferred minor resistance to oxidative stress–induced cell death (12.6 percentage point decrease, P = 0.10), and modestly increased oxygen consumption (36%, P = 0.09). No beneficial effect of the acute or long-term BED serum was observed in old hMPCs. Conclusions In younger women, dietary interventions could be a feasible strategy to improve hMPC function and thus muscle regeneration, through altering the serum environment. This study was registered at clinicaltrials.gov (NCT04262258).

Effects of Graphene Oxide Nanofilm and Chicken Embryo Muscle Extract on Muscle Progenitor Cell Differentiation and Contraction

Finding an effective muscle regeneration technique is a priority for regenerative medicine. It is known that the key factors determining tissue formation include cells, capable of proliferating and/or differentiating, a niche (surface) allowing their colonization and growth factors. The interaction between these factors, especially between the surface of the artificial niche and growth factors, is not entirely clear. Moreover, it seems that the use of a complex of complementary growth factors instead of a few strictly defined ones could increase the effectiveness of tissue maturation, including muscle tissue. In this study, we evaluated whether graphene oxide (GO) nanofilm, chicken embryo muscle extract (CEME), and GO combined with CEME would affect the differentiation and functional maturation of muscle precursor cells, as well as the ability to spontaneously contract a pseudo-tissue muscle. CEME was extracted on day 18 of embryogenesis. Muscle cells obtained from an 8-day-old chicken embryo limb bud were treated with GO and CEME. Cell morphology and differentiation were observed using different microscopy methods. Cytotoxicity and viability of cells were measured by lactate dehydrogenase and Vybrant Cell Proliferation assays. Gene expression of myogenic regulatory genes was measured by Real-Time PCR. Our results demonstrate that CEME, independent of the culture surface, was the main factor influencing the intense differentiation of muscle progenitor cells. The present results, for the first time, clearly demonstrated that the cultured tissue-like structure was capable of inducing contractions without externally applied impulses. It has been indicated that a small amount of CEME in media (about 1%) allows the culture of pseudo-tissue muscle capable of spontaneous contraction. The study showed that the graphene oxide may be used as a niche for differentiating muscle cells, but the decisive influence on the maturation of muscle tissue, especially muscle contractions, depends on the complexity of the applied growth factors.

Glycolytic and Mitochondrial Metabolism Are Essential for Muscle Progenitor Cell Proliferation and Impacted by Pyruvate Kinase M2 (P08-135-19)

Objectives The metabolic requirements for muscle progenitor cell (MPC), the cell required for skeletal muscle regeneration, are ambiguous. We identified that pyruvate kinase M2 (PKM2), a glycolytic enzyme (canonical) and metabolic regulator (non-canonical) promotes MPC proliferation. Our objective was to determine the essentiality of glycolytic and mitochondrial metabolism for MPC proliferation (Experiment [EXP] 1 and 2), and how PKM2 impacts these metabolic pathways (EXP 3). Methods C2C12 cells, immortalized MPCs, were cultured in growth media (GM, DMEM + 10% fetal bovine serum) for all experiments. EXP 1: MPCs were cultured in glucose free GM supplemented with/without 25 mM glucose or 25 mM galactose for 48 h. EXP 2: MPCs were cultured in GM with mitochondrial inhibitors (500 nM Rotenone or 2 μM Oligomycin) for 72 h. EXP 3: MPCs were cultured in GM and treated for 8 h with 150 μM TEPP-46 or 1.4 μM Shikonin, small molecules that differentially impact canonical and non-canonical PKM2 activity. Cell number was measured with a Celigo. Oxygen consumption (OCR, [i.e., oxidative phosphorylation]) and extracellular acidification rates (ECAR [i.e., glycolysis]) were measured with a Seahorse XF Analyzer. Glucose uptake was measured with a kit. Mitochondria membrane potential and copy number were measured with JC1 dye and qPCR, respectively. Results At 48 h, glucose free media and galactose containing media decreased cell number 8.2-fold (P < 0.001) and 7.7-fold (P < 0.01), compared to cells cultured in glucose containing media. At 72 h, cell number was reduced 16.9-fold by Rotenone (P < 0.001) and 14.1-fold by Oligomycin (P < 0.01), compared to vehicle control. Both TEPP-46 and Shikonin increased ECAR (1.6- and 1.4-fold, P < 0.01) while there was a trend toward reduced glucose uptake (1.2-fold and 1.3-fold, P < 0.1). Both TEPP-46 and Shikonin reduced basal OCR (1.6- and 1.3-fold, P < 0.01) and mitochondria membrane potential (1.7- and 1.2-fold, P < 0.01), but did not affect mitochondrial copy number (P > 0.05). Conclusions Both glycolytic and mitochondrial metabolism are essential for MPC proliferation and PKM2 may regulate MPC proliferation through these pathways. Future studies will elucidate the relationship between metabolism, PKM2, and MPC proliferation. Funding Sources NSF Graduate Research Fellowship.