The history: Myelomeningocele - also known as spina bifida- is a devastating congenital anomaly of the central nervoussystem that is caused by the malformation of the spinal cordand vertebral column during embryogenes...The history: Myelomeningocele - also known as spina bifida- is a devastating congenital anomaly of the central nervoussystem that is caused by the malformation of the spinal cordand vertebral column during embryogenesis. Depending onthe location of the spina bifida lesion on the spine, patientssuffer from neurological dysfunction ranging from paresisand incontinence to complete paralysis. The current standardof care for spina bifida is in utero surgical repair of the defect,which has been shown to minimize the secondary deficits associatedwith this disorder (Adzick et al., 2011). Despite thesesuccesses, this approach does not reliably improve neurologicfunction of affected children. Several groups, including ourown, have performed studies aimed at augmenting the inutero surgical repair of spina bifida by applying principlesof stem cell and tissue engineering to provide an enhancedprotection of the exposed neural elements (Saadai et al., 2011,2013; Wang et al., 2015; Brown et al., 2016). The ultimategoal of these studies is to improve the neurologic function inpatients while maintaining the benefits of the existing fetalsurgical treatment.展开更多
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...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.展开更多
基金supported by NIH(No.5R01NS100761-02,5R03HD091601-02)Shriners Hospital for Children research grants(No.87410-NCA-17 and 85119-NCA-18)March of Dimes Foundation(No.5FY1682)to AW
文摘The history: Myelomeningocele - also known as spina bifida- is a devastating congenital anomaly of the central nervoussystem that is caused by the malformation of the spinal cordand vertebral column during embryogenesis. Depending onthe location of the spina bifida lesion on the spine, patientssuffer from neurological dysfunction ranging from paresisand incontinence to complete paralysis. The current standardof care for spina bifida is in utero surgical repair of the defect,which has been shown to minimize the secondary deficits associatedwith this disorder (Adzick et al., 2011). Despite thesesuccesses, this approach does not reliably improve neurologicfunction of affected children. Several groups, including ourown, have performed studies aimed at augmenting the inutero surgical repair of spina bifida by applying principlesof stem cell and tissue engineering to provide an enhancedprotection of the exposed neural elements (Saadai et al., 2011,2013; Wang et al., 2015; Brown et al., 2016). The ultimategoal of these studies is to improve the neurologic function inpatients while maintaining the benefits of the existing fetalsurgical treatment.
文摘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.