Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Transplantation of committed pre-adipocytes from brown adipose tissue improves whole-body glucose homeostasis.

Obesity and its co-morbidities including type 2 diabetes are increasing at epidemic rates in the U.S. and worldwide. Brown adipose tissue (BAT) is a potential therapeutic to combat obesity and type 2 diabetes. Increasing BAT mass by transplantation improves metabolic health in rodents, but its clinical translation remains a challenge. Here, we investigated if transplantation of 2-4 million differentiated brown pre-adipocytes from mouse BAT stromal fraction (SVF) or human pluripotent stem cells (hPSCs) could improve metabolic health. Transplantation of differentiated brown pre-adipocytes, termed "committed pre-adipocytes" from BAT SVF from mice or derived from hPSCs improves glucose homeostasis and insulin sensitivity in recipient mice under conditions of diet-induced obesity, and this improvement is mediated through the collaborative actions of the liver transcriptome, tissue AKT signaling, and FGF21. These data demonstrate that transplantation of a small number of brown adipocytes has significant long-term translational and therapeutic potential to improve glucose metabolism.

Laminin α4 Expression in Human Adipose Tissue Depots and Its Association with Obesity and Obesity Related Traits.

Laminin α4 (LAMA4) is one of the main structural adipocyte basement membrane (BM) components that is upregulated during adipogenesis and related to obesity in mice and humans. We conducted RNA-seq-based gene expression analysis of LAMA4 in abdominal subcutaneous (SC) and visceral (VIS) adipose tissue (AT) depots across three human sub-cohorts of the Leipzig Obesity BioBank (LOBB) to explore the relationship between LAMA4 expression and obesity (N = 1479) in the context of weight loss (N = 65) and metabolic health (N = 42). We found significant associations of LAMA4 with body fat mass (p < 0.001) in VIS AT; higher expression in VIS AT compared to SC AT; and significant relation to metabolic health parameters e.g., body fat in VIS AT, waist (p = 0.009) and interleukin 6 (p = 0.002) in male VIS AT, and hemoglobin A1c (p = 0.008) in male SC AT. AT LAMA4 expression was not significantly different between subjects with or without obesity, metabolically healthy versus unhealthy, and obesity before versus after short-term weight loss. Our results support significant associations between obesity related clinical parameters and elevated LAMA4 expression in humans. Our work offers one of the first references for understanding the meaning of LAMA4 expression specifically in relation to obesity based on large-scale RNA-seq data.

Transgene-free direct conversion of murine fibroblasts into functional muscle stem cells.

Transcription factor-based cellular reprogramming provides an attractive approach to produce desired cell types for regenerative medicine purposes. Such cellular conversions are widely dependent on viral vectors to efficiently deliver and express defined factors in target cells. However, use of viral vectors is associated with unfavorable genomic integrations that can trigger deleterious molecular consequences, rendering this method a potential impediment to clinical applications. Here, we report on a highly efficient transgene-free approach to directly convert mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by overexpression of synthetic MyoD-mRNA in concert with an enhanced small molecule cocktail. First, we performed a candidate compound screen and identified two molecules that enhance fibroblast reprogramming into iMPCs by suppression of the JNK and JAK/STAT pathways. Simultaneously, we developed an optimal transfection protocol to transiently overexpress synthetic MyoD-mRNA in fibroblasts. Combining these two techniques enabled robust and rapid reprogramming of fibroblasts into Pax7 positive iMPCs in as little as 10 days. Nascent transgene-free iMPCs proliferated extensively in vitro, expressed a suite of myogenic stem cell markers, and could differentiate into highly multinucleated and contractile myotubes. Furthermore, using global and single-cell transcriptome assays, we delineated gene expression changes associated with JNK and JAK/STAT pathway inhibition during reprogramming, and identified in iMPCs a Pax7+ stem cell subpopulation resembling satellite cells. Last, transgene-free iMPCs robustly engrafted skeletal muscles of a Duchenne muscular dystrophy mouse model, restoring dystrophin expression in hundreds of myofibers. In summary, this study reports on an improved and clinically safer approach to convert fibroblasts into myogenic stem cells that can efficiently contribute to muscle regeneration in vivo.

Monoallelic intragenic POU3F2 variants lead to neurodevelopmental delay and hyperphagic obesity, confirming the gene's candidacy in 6q16.1 deletions.

While common obesity accounts for an increasing global health burden, its monogenic forms have taught us underlying mechanisms via more than 20 single-gene disorders. Among these, the most common mechanism is central nervous system dysregulation of food intake and satiety, often accompanied by neurodevelopmental delay (NDD) and autism spectrum disorder. In a family with syndromic obesity, we identified a monoallelic truncating variant in POU3F2 (alias BRN2) encoding a neural transcription factor, which has previously been suggested as a driver of obesity and NDD in individuals with the 6q16.1 deletion. In an international collaboration, we identified ultra-rare truncating and missense variants in another ten individuals sharing autism spectrum disorder, NDD, and adolescent-onset obesity. Affected individuals presented with low-to-normal birth weight and infantile feeding difficulties but developed insulin resistance and hyperphagia during childhood. Except for a variant leading to early truncation of the protein, identified variants showed adequate nuclear translocation but overall disturbed DNA-binding ability and promotor activation. In a cohort with common non-syndromic obesity, we independently observed a negative correlation of POU3F2 gene expression with BMI, suggesting a role beyond monogenic obesity. In summary, we propose deleterious intragenic variants of POU3F2 to cause transcriptional dysregulation associated with hyperphagic obesity of adolescent onset with variable NDD.

Genetic variants in genes involved in creatine biosynthesis in patients with severe obesity or anorexia nervosa.

Increased thermogenesis in brown adipose tissue might have an obesity-reducing effect in humans. In transgenic mice, depletion of genes involved in creatine metabolism results in disrupted thermogenic capacity and altered effects of high-fat feeding on body weight. Data analyses of a sex-stratified genome-wide association study (GWAS) for body mass index (BMI) within the genomic regions of genes of this pathway (CKB, CKMT1B, and GATM) revealed one sex-dimorphic BMI-associated SNP in CKB (rs1136165). The effect size was larger in females than in males. A mutation screen of the coding regions of these three candidate genes in a screening group (192 children and adolescents with severe obesity, 192 female patients with anorexia nervosa, and 192 healthy-lean controls) identified five variants in each, CKB and GATM, and nine variants in the coding sequence of CKMT1B. Non-synonymous variants identified in CKB and CKMT1B were genotyped in an independent confirmation study group (781 families with severe obesity (trios), 320 children and adolescents with severe obesity, and 253 healthy-lean controls). In silico tools predicted mainly benign yet protein-destabilizing potentials. A transmission disequilibrium test in trios with severe obesity indicated an obesity-protective effect of the infrequent allele at rs149544188 located in CKMT1B. Subsequent correlation analyses in 1,479 individuals of the Leipzig Obesity BioBank revealed distinct correlations of CKB with the other two genes in omental visceral adipose tissue (VAT) and abdominal subcutaneous adipose tissue (SAT). Furthermore, between-subject comparisons of gene expression levels showed generally higher expressions of all three genes of interest in VAT than in SAT. Future in vitro analyses are needed to assess the functional implications of these findings.

Obesity Is Associated with Distorted Proteoglycan Expression in Adipose Tissue.

Proteoglycans are central components of the extracellular matrix (ECM) and binding partners for inflammatory chemokines. Morphological differences in the ECM and increased inflammation are prominent features of the white adipose tissues in patients with obesity. The impact of obesity and weight loss on the expression of specific proteoglycans in adipose tissue is not well known. This study aimed to investigate the relationship between adiposity and proteoglycan expression. We analyzed transcriptomic data from two human bariatric surgery cohorts. In addition, RT-qPCR was performed on adipose tissues from female and male mice fed a high-fat diet. Both visceral and subcutaneous adipose tissue depots were analyzed. Adipose mRNA expression of specific proteoglycans, proteoglycan biosynthetic enzymes, proteoglycan partner molecules, and other ECM-related proteins were altered in both human cohorts. We consistently observed more profound alterations in gene expression of ECM targets in the visceral adipose tissues after surgery (among others VCAN (p = 0.000309), OGN (p = 0.000976), GPC4 (p = 0.00525), COL1A1 (p = 0.00221)). Further, gene analyses in mice revealed sex differences in these two tissue compartments in obese mice. We suggest that adipose tissue repair is still in progress long after surgery, which may reflect challenges in remodeling increased adipose tissues. This study can provide the basis for more mechanistic studies on the role of proteoglycans in adipose tissues in obesity.

Exclusive generation of rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells.

Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.

On-resin N-terminal peptoid degradation: Toward mild sequencing conditions.

A novel approach to sequentially degrade peptoid N-terminal N-(substituted)glycine residues on the solid-phase using very mild conditions is reported. This method relies on the treatment of resin-bound, bromoacetylated peptoids with silver perchlorate in THF, leading to an intramolecular cyclization reaction to liberate the terminal residue as a N-substituted morpholine-2,5-dione, resulting in a truncated peptoid upon hydrolysis and a silver bromide byproduct. Side-chain functional group tolerance is explored and reaction kinetics are determined. In a series of pentapeptoids possessing variable, non-nucleophilic side-chains at the second position (R(2) ), we demonstrate that sequential N-terminal degradation of the first two residues proceeds in 87% and 74% conversions on average, respectively. We further demonstrate that the degradation reaction is selective for peptoids, and represents substantial progress toward a mild, iterative sequencing method for peptoid oligomers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 726-736, 2016.