Lymphoid reconstitution of the human fetal thymus in SCID mice with CD34+ precursor cells

The search for human hematopoietic stem cells has been hampered by the lack of appropriate assay systems. Demonstration of the ability of precursor cell candidates to give rise to T cells is of significant difficulty since dissociated in vitro cultured thymus stroma cells lose their ability to sustain thymocyte maturation. To define further the differentiative capacities of the rare human fetal liver and bone marrow cells that express the CD34 surface antigen and exhibit in vitro myeloid and pre-B cell activities, we have microinjected them into HLA-mismatched fetal thymus fragments, partially depleted of hematopoietic cells by low temperature culture. In vitro colonized thymuses have then been allowed to develop upon engraftment into immunodeficient SCID mice. Using this modification of the SCID-hu system, we show that low numbers of fetal CD34+ progenitor cells can repopulate the lymphoid compartment in the human thymus

Emergence of human angiohematopoietic cells in normal development and from cultured embryonic stem cells

Human hematopoiesis proceeds transiently in the extraembryonic yolk sac and embryonic, then fetal liver before being stabilized in the bone marrow during the third month of gestation. In addition to this classic developmental sequence, we have previously shown that the aorta-gonad-mesonephros (AGM) embryonic territory produces stem cells for definitive hematopoiesis from 27 to 40 days of human development, through an intermediate blood-forming endothelium stage. These studies have relied on the use of traditional markers of human hematopoietic and endothelial cells. In addition, we have recently identified and characterized a novel surface molecule, BB9, which typifies the earliest founders of the human angiohematopoietic system. BB9, which was initially identified with a monoclonal antibody raised to Stro-1(+) bone marrow stromal cells, recognizes in the adult the most primitive Thy-1(+) CD133(+) Lin(−), non-obese diabetic—severe combined immunodeficiency disease (NOD–SCID) mouse engrating hematopoietic stem cells (HSCs). In the 3- to 4-week embryo,BB9expression typifies a subset of splanchnopleural mesodermal cells that migrate dorsally and colonize the ventral aspect of the aorta where they establish a population of hemogenic endothelial cells. We have indeed confirmed that hematopoietic potential in the human embryo, as assessed by long-term culture-initiating cell (LTC-IC) and SCID mouse reconstituting cell (SRC) activities, is confined to BB9-expressing cells. We have further validated these results in the model of human embryonic stem cells (hESCs) in which we have modeled, through the development of hematopoietic embryoid bodies (EBs), primitive and definitive hematopoieses. In this setting, we have documented the emergence of BB9(+) hemangioblast-like clonogenic angiohematopoietic progenitors that currently represent the earliest known founders of the human vascular and blood systems

Human airway xenograft models of epithelial cell regeneration

Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa

Electrical Stimulation Influences Satellite Cell Proliferation and Apoptosis in Unloading-Induced Muscle Atrophy in Mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention

Myoblast sensitivity and fibroblast insensitivity to osteogenic conversion by BMP-2 correlates with the expression of Bmpr-1a

<p>Abstract</p> <p>Background</p> <p>Osteoblasts are considered to primarily arise from osseous progenitors within the periosteum or bone marrow. We have speculated that cells from local soft tissues may also take on an osteogenic phenotype. Myoblasts are known to adopt a bone gene program upon treatment with the osteogenic bone morphogenetic proteins (BMP-2,-4,-6,-7,-9), but their osteogenic capacity relative to other progenitor types is unclear. We further hypothesized that the sensitivity of cells to BMP-2 would correlate with BMP receptor expression.</p> <p>Methods</p> <p>We directly compared the BMP-2 sensitivity of myoblastic murine cell lines and primary cells with osteoprogenitors from osseous tissues and fibroblasts. Fibroblasts forced to undergo myogenic conversion by transduction with a MyoD-expressing lentiviral vector (LV-MyoD) were also examined. Outcome measures included alkaline phosphatase expression, matrix mineralization, and expression of osteogenic genes <it>(alkaline phosphatase, osteocalcin </it>and <it>bone morphogenetic protein receptor-1A) </it>as measured by quantitative PCR.</p> <p>Results</p> <p>BMP-2 induced a rapid and robust osteogenic response in myoblasts and osteoprogenitors, but not in fibroblasts. Myoblasts and osteoprogenitors grown in osteogenic media rapidly upregulated <it>Bmpr-1a </it>expression. Chronic BMP-2 treatment resulted in peak <it>Bmpr-1a </it>expression at day 6 before declining, suggestive of a negative feedback mechanism. In contrast, fibroblasts expressed low levels of <it>Bmpr-1a </it>that was only weakly up-regulated by BMP-2 treatment. Bioinformatics analysis confirmed the presence of myogenic responsive elements in the proximal promoter region of human and murine <it>BMPR-1A/Bmpr-1a</it>. Forced myogenic gene expression in fibroblasts was associated with a significant increase in <it>Bmpr-1a </it>expression and a synergistic increase in the osteogenic response to BMP-2.</p> <p>Conclusion</p> <p>These data demonstrate the osteogenic sensitivity of muscle progenitors and provide a mechanistic insight into the variable response of different cell lineages to BMP-2.</p

Platelet-Rich Plasma Promotes the Proliferation of Human Muscle Derived Progenitor Cells and Maintains Their Stemness

Human muscle-derived progenitor cells (hMDPCs) offer great promise for muscle cell-based regenerative medicine; however, prolonged ex-vivo expansion using animal sera is necessary to acquire sufficient cells for transplantation. Due to the risks associated with the use of animal sera, the development of a strategy for the ex vivo expansion of hMDPCs is required. The purpose of this study was to investigate the efficacy of using platelet-rich plasma (PRP) for the ex-vivo expansion of hMDPCs. Pre-plated MDPCs, myoendothelial cells, and pericytes are three populations of hMDPCs that we isolated by the modified pre-plate technique and Fluorescence Activated Cell Sorting (FACS), respectively. Pooled allogeneic human PRP was obtained from a local blood bank, and the effect that thrombin-activated PRP-releasate supplemented media had on the ex-vivo expansion of the hMDPCs was tested against FBS supplemented media, both in vitro and in vivo. PRP significantly enhanced short and long-term cell proliferation, with or without FBS supplementation. Antibody-neutralization of PDGF significantly blocked the mitogenic/proliferative effects that PRP had on the hMDPCs. A more stable and sustained expression of markers associated with stemness, and a decreased expression of lineage specific markers was observed in the PRP-expanded cells when compared with the FBS-expanded cells. The in vitro osteogenic, chondrogenic, and myogenic differentiation capacities of the hMDPCs were not altered when expanded in media supplemented with PRP. All populations of hMDPCs that were expanded in PRP supplemented media retained their ability to regenerate myofibers in vivo. Our data demonstrated that PRP promoted the proliferation and maintained the multi-differentiation capacities of the hMDPCs during ex-vivo expansion by maintaining the cells in an undifferentiated state. Moreover, PDGF appears to be a key contributing factor to the beneficial effect that PRP has on the proliferation of hMDPCs. © 2013 Li et al

A Novel Conserved Isoform of the Ubiquitin Ligase UFD2a/UBE4B Is Expressed Exclusively in Mature Striated Muscle Cells

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3′ introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis

Differential Requirement for Utrophin in the Induced Pluripotent Stem Cell Correction of Muscle versus Fat in Muscular Dystrophy Mice

Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle

Relationships between Hematopoiesis and Hepatogenesis in the Midtrimester Fetal Liver Characterized by Dynamic Transcriptomic and Proteomic Profiles

In fetal hematopoietic organs, the switch from hematopoiesis is hypothesized to be a critical time point for organogenesis, but it is not yet evidenced. The transient coexistence of hematopoiesis will be useful to understand the development of fetal liver (FL) around this time and its relationship to hematopoiesis. Here, the temporal and the comparative transcriptomic and proteomic profiles were observed during the critical time points corresponding to the initiation (E11.5), peak (E14.5), recession (E15.5), and disappearance (3 ddp) of mouse FL hematopoiesis. We found that E11.5-E14.5 corresponds to a FL hematopoietic expansion phase with distinct molecular features, including the expression of new transcription factors, many of which are novel KRAB (Kruppel-associated box)-containing zinc finger proteins. This time period is also characterized by extensive depression of some liver functions, especially catabolism/utilization, immune and defense, classical complement cascades, and intrinsic blood coagulation. Instead, the other liver functions increased, such as xenobiotic and sterol metabolism, synthesis of carbohydrate and glycan, the alternate and lectin complement cascades and extrinsic blood coagulation, and etc. Strikingly, all of the liver functions were significantly increased at E14.5-E15.5 and thereafter, and the depression of the key pathways attributes to build the hematopoietic microenvironment. These findings signal hematopoiesis emigration is the key to open the door of liver maturation