Clonal isolation of endothelial colony-forming cells from early gestation chorionic villi of human placenta for fetal tissue regeneration

BACKGROUND Endothelial colony-forming cells(ECFCs)have been implicated in the process of vascularization,which includes vasculogenesis and angiogenesis.Vasculogenesis is a de novo formation of blood vessels,and is an essential physiological process that occurs during embryonic development and tissue regeneration.Angiogenesis is the growth of new capillaries from pre-existing blood vessels,which is observed both prenatally and postnatally.The placenta is an organ composed of a variety of fetal-derived cells,including ECFCs,and therefore has significant potential as a source of fetal ECFCs for tissue engineering.AIM To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi(CV-ECFCs)of the placenta,and assess their potential for tissue engineering.METHODS The early gestation chorionic villus tissue was dissociated by enzyme digestion.Cells expressing CD31 were selected using magnetic-activated cell sorting,and plated in endothelial-specific growth medium.After 2-3 wks in culture,colonies displaying cobblestone-like morphology were manually picked using cloning cylinders.We characterized CV-ECFCs by flow cytometry,immunophenotyping,tube formation assay,and Dil-Ac-LDL uptake assay.Viral transduction of CVECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector,and transduction efficiency was tested by fluorescent microscopy and flow cytometry.Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved,small intestinal submucosa extracellular matrix scaffold.RESULTS After four passages in 6-8 wks of culture,we obtained a total number of 1.8×107 CV-ECFCs using 100 mg of early gestational chorionic villus tissue.Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31,CD144,CD146,CD105,CD309,only partially expressed CD34,and did not express CD45 and CD90.CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation,similar to cord blood-derived ECFCs(CB-ECFCs).CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%.Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold.CONCLUSION In summary,we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs.A substantial number of CV-ECFCs can be obtained within a short time frame,representing a promising novel source of ECFCs for fetal treatments.

Early gestation chorionic villi-derived stromal cells for fetal tissue engineering

AIM: To investigate the potential for early gestation placenta-derived mesenchymal stromal cells(PMSCs) for fetal tissue engineering.METHODS: PMSCs were isolated from early gestation chorionic villus tissue by explant culture. Chorionic villus sampling(CVS)-size tissue samples(mean = 35.93 mg)were used to test the feasibility of obtaining large cell numbers from CVS within a clinically relevant timeframe. We characterized PMSCs isolated from 6 donor placentas by flow cytometry immunophenotyping, multipotency assays, and through immunofluorescent staining. Protein secretion from PMSCs was examined using two cytokine array assays capable of probing for over 70 factors in total. Delivery vehicle compatibility of PMSCs was determined using three common scaffold systems: fibrin glue, collagen hydrogel, and biodegradable nanofibrous scaffolds made from a combination of polylactic acid(PLA) and poly(lactic-co-glycolic acid)(PLGA). Viral transduction of PMSCs was performed using a Luciferase-GFPcontaining lentiviral vector and efficiency of transduction was tested by fluorescent microscopy and flow cytometry analysis.RESULTS: We determined that an average of 2.09 × 106(SD ± 8.59 × 105) PMSCs could be obtained from CVS-size tissue samples within 30 d(mean = 27 d, SD ± 2.28), indicating that therapeutic numbers of cells can be rapidly expanded from very limited masses of tissue. Immunophenotyping by flow cytometry demonstrated that PMSCs were positive for MSC markers CD105, CD90, CD73, CD44, and CD29, and were negative for hematopoietic and endothelial markers CD45, CD34, and CD31. PMSCs displayed trilineage differentiation capability, and were found to express developmental transcription factors Sox10 and Sox17 as well as neuralrelated structural proteins NFM, Nestin, and S100 β. Cytokine arrays revealed a robust and extensive profile of PMSC-secreted cytokines and growth factors, and detected 34 factors with spot density values exceeding 103. Detected factors had widely diverse functions that include modulation of angiogenesis and immune response, cell chemotaxis, cell proliferation, blood vessel maturation and homeostasis, modulation of insulin-like growth factor activity, neuroprotection, extracellular matrix degradation and even blood coagulation. Importantly, PMSCs were also determined to be compatible with bothbiological and synthetic material-based delivery vehicles such as collagen and fibrin hydrogels, and biodegradable nanofiber scaffolds made from a combination of PLA and PLGA. Finally, we demonstrated that PMSCs can be efficiently transduced(> 95%) with a Luciferase-GFPcontaining lentiviral vector for future in vivo cell tracking after transplantation.CONCLUSION: Our findings indicate that PMSCs represent a unique source of cells that can be effectively utilized for in utero cell therapy and tissue engineering.

胎儿外科 正在证明可用于几种新生儿疾病的治疗

出生前确诊的大多数疾病最好等到出生后再做处理。然而,对预计可能影响发育的少数疾病,在胎儿期就进行治疗是有好处的。在美国,胎儿外科手术主要在两个医疗中心已经开展了20多年。近来,胎儿外科已经成为一种全球性的尝试,全世界有将近12个中心开展这项工作。研究至今,仍然认为只有存在威胁胎儿生命的缺陷才考虑在出生前进行矫正。目前。