PitViper: a software for comparative meta-analysis and annotation of functional screening data.

Recent advancements in shRNA and Cas protein technologies have enabled functional screening methods targeting genes or non-coding regions using single or pooled shRNA and sgRNA. CRISPR-based systems have also been developed for modulating DNA accessibility, resulting in CRISPR-mediated interference (CRISPRi) or activation (CRISPRa) of targeted genes or genomic DNA elements. However, there is still a lack of software tools for integrating diverse array of functional genomics screening outputs that could offer a cohesive framework for comprehensive data integration. Here, we developed PitViper, a flexible and interactive open-source software designed to fill this gap, providing reliable results for the type of elements being screened. It is an end-to-end automated and reproducible bioinformatics pipeline integrating gold-standard methods for functional screening analysis. Our sensitivity analyses demonstrate that PitViper is a useful tool for identifying potential super-enhancer liabilities in a leukemia cell line through genome-wide CRISPRi-based screening. It offers a robust, flexible, and interactive solution for integrating data analysis and reanalysis from functional screening methods, making it a valuable resource for researchers in the field.

Screening of ETO2-GLIS2-induced Super Enhancers identifies targetable cooperative dependencies in acute megakaryoblastic leukemia.

Super Enhancers (SEs) are clusters of regulatory elements associated with cell identity and disease. However, whether these elements are induced by oncogenes and can regulate gene modules cooperating for cancer cell transformation or maintenance remains elusive. To address this question, we conducted a genome-wide CRISPRi-based screening of SEs in ETO2-GLIS2+ acute megakaryoblastic leukemia. This approach revealed SEs essential for leukemic cell growth and survival that are induced by ETO2-GLIS2 expression. In particular, we identified a de novo SE specific of this leukemia subtype and regulating expression of tyrosine kinase-associated receptors KIT and PDGFRA. Combined expression of these two receptors was required for leukemic cell growth, and CRISPRi-mediated inhibition of this SE or treatment with tyrosine kinase inhibitors impaired progression of leukemia in vivo in patient-derived xenografts experiments. Our results show that fusion oncogenes, such as ETO2-GLIS2, can induce activation of SEs regulating essential gene modules synergizing for leukemia progression.

Human Aortic Valve Interstitial Cells Display Proangiogenic Properties During Calcific Aortic Valve Disease.

OBJECTIVE: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90+/CD73+/CD44+ expression and multipotent-like differentiation ability. When VICp were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VICp-conditioned media and confirmed at the mRNA level in VICp compared with control VIC. Conditioned media from VICp induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in angiogenesis by a VEGF-A dependent mechanism. CONCLUSIONS: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.

Valproic Acid Decreases Endothelial Colony Forming Cells Differentiation and Induces Endothelial-to-Mesenchymal Transition-like Process.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor is a widely used anticonvulsant drug. VPA is also under clinical evaluation to be employed in anticancer therapy, as an antithrombotic agent or a molecule to be used in the stem cells expansion protocols. Since endothelial colony forming cells (ECFC) has been identified as the human postnatal vasculogenic cells involved in thrombotic disorders and serve as a promising source of immature cell for vascular repair, objectives of the present study were to determine how VPA contributes to ECFC commitment and their angiogenic properties. We examined the effect of VPA on ECFC obtained from cord blood by evaluating colony number, proliferation, migration and their sprouting ability in vitro, as well as their in vivo vasculogenic properties. VPA inhibited endothelial differentiation potential from of cord blood derived stem cells associated with decreased proliferation and sprouting activity of cultured ECFC. VPA treatment significantly decreased the vessel-forming ability of ECFC transplanted together with mesenchymal stem cells (MSC) in Matrigel implants in nude mice model. Surprisingly, a microscopic evaluation revealed that VPA induces marked morphological changes from a cobblestone-like EC morphology to enlarged spindle shaped morphology of ECFC. RT-qPCR and a CD31/CD90 flow cytometry analysis confirmed a phenotypic switch of VPA-treated ECFC to mesenchymal-like phenotype. In conclusion, the pan-HDAC inhibitor VPA described for expansion of hematopoietic stem cells and very small embryonic like stem cells cannot be successfully employed for differentiation of endothelial lineage committed ECFC into functional endothelial cells. Our data also suggest that VPA based therapeutics may induce endothelial dysfunction associated with fibrosis that might induce thrombosis recurrence or venous insufficiency.

Interleukin-8 release by endothelial colony-forming cells isolated from idiopathic pulmonary fibrosis patients might contribute to their pathogenicity.

INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by obliteration of alveolar architecture, resulting in declining lung function and ultimately death. Pathogenic mechanisms involve a concomitant accumulation of scar tissue together with myofibroblasts activation and a strong abnormal vascular remodeling. Endothelial progenitor cells (ECFC subtype) have been investigated in several human lung diseases as a potential actor in IPF. We previously demonstrated that ECFCs are down-regulated in IPF in contrast to healthy controls. We postulated here that ECFCs might behave as a liquid biopsy in IPF patients and that they exert modified vasculogenic properties. METHODS AND RESULTS: ECFCs isolated from controls and IPF patients expressed markers of the endothelial lineage and did not differ concerning adhesion, migration, and differentiation in vitro and in vivo. However, senescent and apoptotic states were increased in ECFCs from IPF patients as shown by galactosidase staining, p16 expression, and annexin-V staining. Furthermore, conditioned medium of IPF-ECFCs had increased level of interleukin-8 that induced migration of neutrophils in vitro and in vivo. In addition, an infiltration by neutrophils was shown in IPF lung biopsies and we found in a prospective clinical study that a high level of neutrophils in peripheral blood of IPF patients was associated to a poor prognosis. CONCLUSION: To conclude, our study shows that IPF patients have a senescent ECFC phenotype associated with an increased IL-8 secretion potential that might contribute to lung neutrophils invasion during IPF.

Endothelial Microparticles are Associated to Pathogenesis of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by obliteration of alveolar architecture, resulting in declining lung function and ultimately death. Pathogenic mechanisms remain unclear but involve a concomitant accumulation of scar tissue together with myofibroblasts activation. Microparticles (MPs) have been investigated in several human lung diseases as possible pathogenic elements, prognosis markers and therapeutic targets. We postulated that levels and cellular origins of circulating MPs might serve as biomarkers in IPF patients and/or as active players of fibrogenesis. Flow cytometry analysis showed a higher level of Annexin-V positive endothelial and platelet MPs in 41 IPF patients compared to 22 healthy volunteers. Moreover, in IPF patients with a low diffusing capacity of the lung for carbon monoxide (DLCO<40%), endothelial MPs (EMPs) were found significantly higher compared to those with DLCO>40% (p = 0.02). We then used EMPs isolated from endothelial progenitor cells (ECFCs) extracted from IPF patients or controls to modulate normal human lung fibroblast (NHLF) properties. We showed that EMPs did not modify proliferation, collagen deposition and myofibroblast transdifferentiation. However, EMPs from IPF patients stimulated migration capacity of NHLF. We hypothesized that this effect could result from EMPs fibrinolytic properties and found indeed higher plasminogen activation potential in total circulating MPs and ECFCs derived MPs issued from IPF patients compared to those isolated from healthy controls MPs. Our study showed that IPF is associated with an increased level of EMPs in the most severe patients, highlighting an active process of endothelial activation in the latter. Endothelial microparticles might contribute to the lung fibroblast invasion mediated, at least in part, by a fibrinolytic activity.

Egfl7 Represses the Vasculogenic Potential of Human Endothelial Progenitor Cells.

Egfl7 (VE-statin) is a secreted protein mostly specific to the endothelial lineage during development and in the adult and which expression is enhanced during angiogenesis. Egfl7 involvement in human postnatal vasculogenesis remains unresolved yet. Our aim was to assess Egfl7 expression in several angiogenic cell types originating from human bone marrow, peripheral blood, or cord blood. We found that only endothelial colony forming cells (ECFC), which are currently considered as the genuine endothelial precursor cells, expressed large amounts of Egfl7. In order to assess its potential roles in ECFC, Egfl7 was repressed in ECFC by RNA interference and ECFC angiogenic capacities were tested in vitro and in vivo. Cell proliferation, differentiation, and migration were significantly improved when Egfl7 was repressed in ECFC in vitro, whereas miR-126-3p levels remained unchanged. In vivo, repression of Egfl7 in ECFC significantly improved post-ischemic revascularization in a model of mouse hind-limb ischemia. In conclusion, ECFC are the sole postnatal angiogenic cells which express large amounts of Egfl7 and whose angiogenic properties are repressed by this factor. Thus, Egfl7 inhibition may be considered as a therapeutic option to improve ECFC-mediated postnatal vasculogenesis and to optimize in vitro ECFC expansion in order to develop an optimized cell therapy approach.

IGF-1 contributes to the expansion of melanoma-initiating cells through an epithelial-mesenchymal transition process.

Melanoma is a particularly virulent human cancer, due to its resistance to conventional treatments and high frequency of metastasis. Melanomas contain a fraction of cells, the melanoma-initiating cells (MICs), responsible for tumor propagation and relapse. Identification of the molecular pathways supporting MICs is, therefore, vital for the development of targeted treatments. One factor produced by melanoma cells and their microenvironment, insulin-like growth factor-1 (IGF- 1), is linked to epithelial-mesenchymal transition (EMT) and stemness features in several cancers.We evaluated the effect of IGF-1 on the phenotype and chemoresistance of B16-F10 cells. IGF-1 inhibition in these cells prevented malignant cell proliferation, migration and invasion, and lung colony formation in immunodeficient mice. IGF-1 downregulation also markedly inhibited EMT, with low levels of ZEB1 and mesenchymal markers (N-cadherin, CD44, CD29, CD105) associated with high levels of E-cadherin and MITF, the major regulator of melanocyte differentiation. IGF-1 inhibition greatly reduced stemness features, including the expression of key stem markers (SOX2, Oct-3/4, CD24 and CD133), and the functional characteristics of MICs (melanosphere formation, aldehyde dehydrogenase activity, side population). These features were associated with a high degree of sensitivity to mitoxantrone treatment.In this study, we deciphered new connections between IGF-1 and stemness features and identified IGF-1 as instrumental for maintaining the MIC phenotype. The IGF1/IGF1-R nexus could be targeted for the development of more efficient anti-melanoma treatments. Blocking the IGF-1 pathway would improve the immune response, decrease the metastatic potential of tumor cells and sensitize melanoma cells to conventional treatments.

[Not Available]. / Utilisation des Cellules Souches Mésenchymateuses en transplantation d'organe.

In solid organ transplant, immunosuppressive therapy helped to increase graft and patient survival. However, these treatments are associated with toxic risks and infectious or tumor complications. The identification of immunoregulatory properties of regulatory cells and in particular Mesenchymal Stem Cells opens new therapeutic perspectives in the prevention of acute rejection and for the treatment of chronic rejection. In this review we will describe immunoregulatory properties of these cells and their potential use in solid organ transplantation.

Human bone marrow mesenchymal stem cells regulate biased DNA segregation in response to cell adhesion asymmetry.

Biased DNA segregation is a mitotic event in which the chromatids carrying the original template DNA strands and those carrying the template copies are not segregated randomly into the two daughter cells. Biased segregation has been observed in several cell types, but not in human mesenchymal stem cells (hMSCs), and the factors affecting this bias have yet to be identified. Here, we have investigated cell adhesion geometries as a potential parameter by plating hMSCs from healthy donors on fibronectin-coated micropatterns. On symmetric micropatterns, the segregation of sister chromatids to the daughter cells appeared random. In contrast, on asymmetric micropatterns, the segregation was biased. This sensitivity to asymmetric extracellular cues was reproducible in cells from all donors but was not observed in human skin-derived fibroblasts or in a fibroblastic cell line used as controls. We conclude that the asymmetry of cell adhesion is a major factor in the regulation of biased DNA segregation in hMSCs.

A prospective study of bone marrow hematopoietic and mesenchymal stem cells in type 1 Gaucher disease patients.

Gaucher disease (GD) is an autosomal recessive disorder characterized by lysosomal glucocerebrosidase (GBA) deficiency leading to hematological and skeletal manifestations. Mechanisms underlying these symptoms have not yet been elucidated. In vivo, bone marrow (BM) mesenchymal stem cells (MSCs) have important role in the regulation of bone mass and in the support of hematopoiesis, thus representing potential candidate that could contribute to the disease. GBA deficiency may also directly impair hematopoietic stem/progenitors cells (HSPCs) intrinsic function and induce hematological defect. In order to evaluate the role of BM stem cells in GD pathophysiology, we prospectively analyzed BM-MSCs and HSPCs properties in a series of 10 patients with type 1 GD. GBA activity was decreased in all tested cell subtypes. GD-MSCs had an impaired growth potential, morphological and cell cycle abnormalities, decreased capacities to differentiate into osteoblasts. Moreover, GD-MSCs secreted soluble factors that stimulated osteoclasts resorbing activities. In vitro and in vivo primitive and mature hematopoiesis were similar between patients and controls. However, GD-MSCs had a lower hematopoietic supportive capacity than those from healthy donors. These data suggest that BM microenvironment is altered in GD and that MSCs are key components of the manifestations observed in GD.

Toll-like receptor 3 regulates cord blood-derived endothelial cell function in vitro and in vivo.

Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties.

In vitro development and characterization of a tissue-engineered conduit resembling esophageal wall using human and pig skeletal myoblast, oral epithelial cells, and biologic scaffolds.

INTRODUCTION: Tissue engineering represents a promising approach for esophageal replacement, considering the complexity and drawbacks of conventional techniques. AIM: To create the components necessary to reconstruct in vitro or in vivo an esophageal wall, we analyzed the feasibility and the optimal conditions of human and pig skeletal myoblast (HSM and PSM) and porcine oral epithelial cell (OEC) culture on biologic scaffolds. MATERIALS AND METHODS: PSM and HSM were isolated from striated muscle and porcine OECs were extracted from oral mucosa biopsies. Myoblasts were seeded on an acellular scaffold issue from porcine small intestinal submucosa (SIS) and OEC on decellularized human amniotic membrane (HAM). Seeding conditions (cell concentrations [0.5×10(6) versus 10(6) cells/cm(2)] and culture periods [7, 14 and 21 days]), were analyzed using the methyl thiazoltetrazolium assay, quantitative PCR, flow cytometry, and immunohistochemistry. RESULTS: Phenotypic stability was observed after cellular expansion for PSM and HSM (85% and 97% CD56-positive cells, respectively), and OECs (90% AE1/AE3- positive cells). After PSM and HSM seeding, quantities of viable cells were similar whatever the initial cell concentration used and remained stable at all time points. During cell culture on SIS, a decrease of CD56-positive cells was observed (76% and 76% by D7, 56% and 70% by D14, 28% and 60% by D21, for PSM and HSM, respectively). Multilayered surface of α-actin smooth muscle and Desmine-positive cells organized in bundles was seen as soon as D7, with no evidence of cell within the SIS. Myoblasts fusion was observed at D21. Pax3 and Pax7 expression was downregulated and MyoD expression upregulated, at D14.OEC proliferation was observed on HAM with both cell concentrations from D7 to D21. The cell metabolism activity was more important on matrix seeded by 10(6) cells/cm(2). With 0.5×10(6) OEC/cm(2), a single layer of pancytokeratin-positive cells was seen at D7, which became pluristratified by D14, while when 10(6) OEC/cm(2) were used, a pluristratified epithelial structure was seen as soon as D7. Proliferative cells (Proliferating Cell Nuclear Antigen staining) were mainly located at the basal layer. CONCLUSION: In this model, the optimal conditions of cell seeding in terms of cell concentration and culture duration were 0.5×10(6) myoblasts/cm(2) and 10(6) OEC/cm(2), and 7 days.

Expression of transforming growth factor ß receptor II in mesenchymal stem cells from systemic sclerosis patients.

OBJECTIVES: The present work aimed to evaluate the expression of transforming growth factor-ß (TGF-ß) receptors on bone marrow-derived multipotent mesenchymal stromal cells (MSCs) in patients with systemic sclerosis (SSc) and the consequences of TGF-ß activation in these cells, since MSC have potential therapeutic interest for SSc patients and knowing that TGF-ß plays a critical role during the development of fibrosis in SSc. DESIGN: This is a prospective research study using MSC samples obtained from SSc patients and compared with MSC from healthy donors. SETTING: One medical hospital involving collaboration between an internal medicine department for initial patient recruitment, a clinical biotherapeutic unit for MSC preparation and an academic laboratory for research. PARTICIPANTS: 9 patients with diffuse SSc for which bone marrow (BM) aspiration was prescribed by sternum aspiration before haematopoietic stem cell transplantation, versus nine healthy donors for normal BM. PRIMARY AND SECONDARY OUTCOME MEASURES: TGF-ß, TGF-ß receptor types I (TBRI) and II (TBRII) mRNA and protein expression were assessed by quantitative PCR and flow cytometry, respectively, in MSC from both SSc patients and healthy donors. MSC were exposed to TGF-ß and assessed for collagen 1α2 synthesis and Smad expression. As positive controls, primary cultures of dermal fibroblasts were also analysed. RESULTS: Compared with nine controls, MSC from nine SSc patients showed significant increase in mRNA levels (p<0.002) and in membrane expression (p<0.0001) of TBRII. In response to TGF-ß activation, a significant increase in collagen 1α synthesis (p<0.05) and Smad-3 phosphorylation was upregulated in SSc MSC. Similar results were obtained on eight SSc-derived dermal fibroblasts compared to six healthy controls. CONCLUSIONS: TBRII gene and protein expression defect in MSC derived from SSc patients may have pathological significance. These findings should be taken into account when considering the use of MSC-based therapies in an autologous setting.

Human Muscle Progenitor Cells Displayed Immunosuppressive Effect through Galectin-1 and Semaphorin-3A.

In human skeletal muscle, myoblasts represent the main population of myogenic progenitors. We previously showed that, beside their myogenic differentiation capacities, myoblasts also differentiate towards osteogenic and chondrogenic lineages, some properties generally considered being hallmarks of mesenchymal stem cells (MSCs). MSCs are also characterized by their immunosuppressive potential, through cell-cell contacts and soluble factors, including prostaglandin E-2 (PGE-2), transforming growth factor-ß1 (TGF-ß1), interleukine-10, or indoleamine 2,3-dioxygenase. We and others also reported that Galectin-1 (Gal-1) and Semaphorin-3A (Sema-3A) were involved in MSCs-mediated immunosuppression. Here, we show that human myoblasts induce a significant and dose-dependant proliferation inhibition, independently of PGE-2 and TGF-ß1. Our experiments revealed that myoblasts, in culture or in situ in human muscles, expressed and secreted Gal-1 and Sema-3A. Furthermore, myoblasts immunosuppressive functions were reverted by using blocking antibodies against Gal-1 or Sema-3A. Together, these results demonstrate an unsuspected immunosuppressive effect of myoblasts that may open new therapeutic perspectives.

Bone marrow microenvironment in an in vitro model of Gaucher disease: consequences of glucocerebrosidase deficiency.

Gaucher disease (GD) is a lysosomal storage disorder due to glucocerebrosidase (GBA) deficiency. Mechanisms leading to the emergence of hematological and skeletal manifestations observed in GD are poorly explained. Bone marrow (BM) mesenchymal stem cells (MSCs) are multipotent progenitors that participate in the regulation of bone mass. MSCs should thus represent a cell population involved in the development or progression of bone disease in GD. In a chemical model of GD obtained with Conduritol ß epoxide (CBE), a specific inhibitor of GBA activity, we functionally characterized BM MSCs and specifically analyzed their capacity to differentiate into osteoblasts. GBA deficiency obtained with CBE treatment, leads to a dramatic impairment of MSCs proliferation and to morphological abnormalities. Although the capacity of MSCs to differentiate into osteoblasts was not modified, the levels of several soluble factors that regulate bone metabolism were increased in MSCs treated with CBE, compared with untreated MSCs. Moreover, addition of conditioned media from CBE-treated MSCs on monocyte-derived osteoclasts cultured on bone matrix leads to an increase of resorption areas. These data suggested that, in GD, MSCs represents a stem cell population that is likely to be involved in bone pathogenesis.

Glucocerebrosidase deficiency dramatically impairs human bone marrow haematopoiesis in an in vitro model of Gaucher disease.

One of the cardinal symptoms of type 1 Gaucher Disease (GD) is cytopenia, usually explained by bone marrow (BM) infiltration by Gaucher cells and hypersplenism. However, some cases of cytopenia in splenectomized or treated patients suggest possible other mechanisms. To evaluate intra-cellular glucocerebrosidase (GlcC) activity in immature progenitors and to prove the conduritol B epoxide (CBE)-induced inhibition of the enzyme, we used an adapted flow cytometric technique before assessing the direct effect of GlcC deficiency in functional assays. Among haematopoietic cells from healthy donors, monocytes showed the highest GlcC activity but immature CD34(+) and mesenchymal cells also had significant GlcC activity. CBE greatly inhibited the enzyme activity of all cell categories. GlcC-deficient CD34(+) cells showed impaired ability to proliferate and differentiate in the expansion assay and had lower frequency of erythroid burst-forming units, granulocyte colony-forming units (CFU) and macrophage CFU progenitors, but the effect of GlcC deficiency on megakaryocyte CFU lineage was not significant. GlcC deficiency strongly impaired primitive haematopoiesis in long-term culture. Furthermore, GlcC deficiency progressively impaired proliferation of mesenchymal progenitors. These data suggest an intrinsic effect of GlcC deficiency on BM immature cells that supplements the pathophysiology of GD and opens new perspectives of therapeutic approach.

In vitro and in vivo analysis of endothelial progenitor cells from cryopreserved umbilical cord blood: are we ready for clinical application?

Umbilical cord blood (CB) represents a main source of circulating endothelial progenitor cells (cEPCs). In view of their clinical use, in either the autologous or allogeneic setting, cEPCs should likely be expanded from CB kept frozen in CB banks. In this study, we compared the expansion, functional features, senescence pattern over culture, and in vivo angiogenic potential of cEPCs isolated from fresh or cryopreserved CB (cryoCB). cEPCs could be isolated in only 59% of cryoCB compared to 94% for fresh CB, while CB units were matched in terms of initial volume, nucleated and CD34(+) cell number. Moreover, the number of endothelial colony-forming cells was significantly decreased when using cryoCB. Once cEPCs culture was established, the proliferation, migration, tube formation, and acetylated-LDL uptake potentials were similar in both groups. In addition, cEPCs derived from cryoCB displayed the same senescence status and telomeres length as that of cEPCs derived from fresh CB. Karyotypic aberrations were found in cells obtained from both fresh and cryoCB. In vivo, in a hind limb ischemia murine model, cEPCs from fresh and cryoCB were equally efficient to induce neovascularization. Thus, cEPCs isolated from cryoCB exhibited similar properties to those of fresh CB in vitro and in vivo. However, the low frequency of cEPCs colony formation after cryopreservation shed light on the need for specific freezing conditions adapted to cEPCs in view of their future clinical use.

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro.

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56(+) cells grew rapidly, a population of CD15(+) cells emerged, partly from CD56(+) cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56(+) and CD15(+) cells shared osteogenic and chondrogenic abilities, while CD56(+) cells presented a myogenic capacity and CD15(+) cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

Esophageal replacement by allogenic aorta in a porcine model.

BACKGROUND: Esophageal replacement is a challenging problem requiring complex reconstruction. In response to the recent success of tracheal replacement by fresh allogenic aorta in humans, we assessed in a pig model the feasibility of circumferential segmental esophageal replacement by a fresh aortic allograft. METHODS: A 4-cm long aortic allograft was interposed after a circumferential 2-cm long resection of the cervical esophagus in 18 minipigs. Anastomoses were protected temporarily by self-expanding polyester-silicone stents (Polyflex; Boston Scientific, Montigny-le-Bretonneux, France). No immunosuppression was given. When stenosis occurred after stent removal or migration, a new stent was inserted. After clinical and endoscopic evaluation, pigs were killed sequentially at 1, 3, 6 and 12 months for analysis. RESULTS: Mortality during the first month was 33%. Four animals died from stent migration during the entire follow-up. Maintenance of a lumen through the graft area by a stent was necessary for 6 months, in order to avoid stenosis occurrence. After the sixth postoperative month, esophageal lumen remained patent until the twelfth month, allowing an apparently normal feeding and weight gain. Gradual contraction of the graft area was observed with time. Sequential histologic analysis showed an inflammatory reaction that decreased with time and a progressive epithelialization of the graft area which became similar to native esophageal epithelium. After 12 months, islets of smooth muscle organized as fascicules or in bundles were visible within the fibrotic tissue. CONCLUSION: Short esophageal replacement by fresh aortic allograft, under the cover of a temporary maintenance of the lumen of the graft area by an esophageal stent, allows the restitution of a patent esophageal lumen and nutritional autonomy.