技术资料

Limited data are available on the effects of stem cells in non-ischemic dilated cardiomyopathy (DCM). Since the diffuse nature of the disease calls for a broad distribution of cells,this study investigated the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) in a mouse model of DCM. Nanofibrous scaffolds were produced using a clinical grade atelocollagen which was electrospun and cross-linked under different conditions. As assessed by scanning electron microscopy and shearwave elastography,the optimum crosslinking conditions for hiPS-CM colonization proved to be a 10% concentration of citric acid crosslinking agent and 150 min of post-electrospinning baking. Acellular collagen scaffolds were first implanted in both healthy mice and those with induced DCM by a cardiac-specific invalidation of serum response factor (SRF). Seven and fourteen days after implantation,the safety of the scaffold was demonstrated by echocardiography and histological assessments. The subsequent step of implantation of the scaffolds seeded with hiPS-CM in DCM induced mice,using cell-free scaffolds as controls,revealed that after fourteen days heart function decreased in controls while it remained stable in the treated mice. This pattern was associated with an increased number of endothelial cells,in line with the greater vascularity of the scaffold. Moreover,a lesser degree of fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling was observed. These results support the interest of the proposed hiPS-CM seeded electrospun scaffold for the stabilization of the DCM outcome with potential for its clinical use in the future. View Publication

Nanofibrous gelatin substrates are suited for long-term expansion of human pluripotent stem cells (hPSCs) under feeder- and serum-free culture conditions. A combinatorial library with different sets of processing parameters was established to assess the culture performance of hPSCs on nanofibrous substrates in terms of cell adhesion and growth rate,using Matrigel as control. Then,the optimal conditions were applied to long-term expansion of hPSCs with several cell lines,showing a maintained pluripotency over more than 20 passages without introducing any abnormal chromosome. In addition,this approach allowed us to avoid enzymatic disassociation and mechanic cutting during passages,thereby promoting a better hPSC culture and long-term expansion. ?? 2014 Elsevier Ltd. View Publication

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling,and differentiation is promoted by BMP signaling,but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium,we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG,OCT4,and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal. View Publication

Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways,which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency,but the precise role and regulation of NANOG are not well defined. View Publication

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8,which is ˜99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD,both have been reported to migrate,on Western blotting (WB),at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore,detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies,immunoprecipitation,mass spectrometry,and studies using recombinant proteins,here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ˜22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate,on denaturing SDS-PAGE,at ˜28 kD to 180 kD. Interestingly,different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins,which can spontaneously form high M.W protein species. Finally,we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether,the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells. View Publication

Inefficient neural differentiation of human induced pluripotent stem cells (hiPSCs) motivates recent investigation of the influence of biophysical characteristics of cellular microenvironment,in particular nanotopography,on hiPSC fate decision. However,the roles of geometry and dimensions of nanotopography in neural lineage commitment of hiPSCs have not been well understood. The objective of this study is to delineate the effects of geometry,feature size and height of nanotopography on neuronal differentiation of hiPSCs. HiPSCs were seeded on equally spaced nanogratings (500 and 1000nm in linewidth) and hexagonally arranged nanopillars (500nm in diameter),each having a height of 150 or 560nm,and induced for neuronal differentiation in concert with dual Smad inhibitors. The gratings of 560nm height reduced cell proliferation,enhanced cytoplasmic localization of Yes-associated protein,and promoted neuronal differentiation (up to 60% βIII-tubulin(+) cells) compared with the flat control. Nanograting-induced cell polarity and cytoplasmic YAP localization were shown to be critical to the induced neural differentiation of hiPSCs. The derived neuronal cells express MAP2,Tau,glutamate,GABA and Islet-1,indicating the existence of multiple neuronal subtypes. This study contributes to the delineation of cell-nanotopography interactions and provides the insights into the design of nanotopography configuration for pluripotent stem cell neural lineage commitment. View Publication

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells,only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study,we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1,a critical transcription factor for pancreatic development,leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore,in the presence of biochemical factors,200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin,glucagon,or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ,suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells. View Publication

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T),cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18),and they displayed the ability to generate NP-like tissue in vitro,which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs,and eventually towards regenerative therapies for disc degeneration. View Publication

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels,differentiation intermediates were investigated. ES cells generated progeny expressing CD34,which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7,the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells,and such lymphoid potential was highly enriched in the CD34+ population. Interestingly,the cytotoxic cells expressed the natural killer (NK) cell markers,such as NKR-P1,perforin,and granzymes,classified into two types,one of which showed target specificity of NK cells. Thus,ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells,and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate,on which VEGF may play an important role. View Publication

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability,the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover,mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein,we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals,including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming,generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically,hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease,showed impaired mitochondrial respiration,being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine. View Publication