RESUMO
We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs), partially reprogrammed cells, somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over â¼10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free, enriched to 95%-99% purity with >80% survival, and had normal transcriptional profiles, differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells.
Assuntos
Adesão Celular , Células-Tronco Pluripotentes/citologia , Diferenciação Celular , Separação Celular , Humanos , CariotipagemRESUMO
INTRODUCTION: Stem cells are being investigated as catalysts of tissue regeneration to either directly replace or promote cellularity lost as a result of traumatic injury or degenerative disease. In many reports, despite low numbers of stably integrated cells, the transient presence of cells delivered or recruited to sites of tissue remodeling globally benefits functional recovery. Such findings have motivated the need to determine how paracrine factors secreted from transplanted cells may be capable of positively impacting endogenous repair processes and somatic cell responses. METHODS: Embryonic stem cells were differentiated as embryoid bodies (EBs) in vitro and media conditioned by EBs were collected at different intervals of time. Gene and protein expression analysis of several different growth factors secreted by EBs were examined by polymerase chain reaction and enzyme-linked immunosorbent assay analysis, respectively, as a function of time. The proliferation and migration of fibroblasts and endothelial cells treated with EB conditioned media was examined compared with unconditioned and growth media controls. RESULTS: The expression of several growth factors, including bone morphogenic protein-4, insulin-like growth factors and vascular endothelial growth factor-A, increased during the course of embryonic stem cell (ESC) differentiation as EBs. Conditioned media collected from EBs at different stages of differentiation stimulated proliferation and migration of both fibroblasts and endothelial cells, based on 5-bromo-2'-deoxyuridine incorporation and transwell assays, respectively. CONCLUSIONS: Overall, these results demonstrate that differentiating ESCs express increasing amounts of various growth factors over time that altogether are capable of stimulating mitogenic and motogenic activity of exogenous cell populations.
Assuntos
Diferenciação Celular , Movimento Celular , Proliferação de Células , Corpos Embrioides/metabolismo , Fibroblastos/fisiologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Células 3T3 , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Meios de Cultivo Condicionados/farmacologia , Corpos Embrioides/citologia , Fator de Crescimento Epidérmico/metabolismo , Fibroblastos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Culturing multipotent adult mesenchymal stem cells as 3D aggregates augments their differentiation potential and paracrine activity. One caveat of stem cell spheroids, though, can be the limited diffusional transport barriers posed by the inherent 3D structure of the multicellular aggregates. In order to circumvent such limitations, polymeric microparticles have been incorporated into stem cell aggregates as a means to locally control the biochemical and physical properties of the 3D microenvironment. However, the introduction of biomaterials to the 3D stem cell microenvironment could alter the mechanical forces sensed by cells within aggregates, which in turn could impact various cell behaviors and overall spheroid mechanics. Therefore, the objective of this study was to determine the acute effects of biomaterial incorporation within mesenchymal stem cell spheroids on aggregate structure and mechanical properties. The results of this study demonstrate that although gelatin microparticle incorporation results in similar multi-cellular organization within human mesenchymal stem cell spheroids, the introduction of gelatin materials significantly impacts spheroid mechanical properties. The marked differences in spheroid mechanics induced by microparticle incorporation may hold major implications for in vitro directed differentiation strategies and offer a novel route to engineer the mechanical properties of tissue constructs ex vivo.
Assuntos
Microambiente Celular/efeitos dos fármacos , Gelatina/farmacologia , Fenômenos Mecânicos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Microesferas , Esferoides Celulares/citologia , Animais , Fenômenos Biomecânicos , Diferenciação Celular/efeitos dos fármacos , Gelatina/química , Humanos , Medicina Regenerativa , Esferoides Celulares/efeitos dos fármacos , Engenharia TecidualRESUMO
The controlled assembly and organization of multi-cellular systems to mimic complex tissue structures is critical to the engineering of tissues for therapeutic and diagnostic applications. Recent advances in micro-scale technologies to control multi-cellular aggregate formation typically require chemical modification of the interface between cells and materials and lack multi-scale flexibility. Here we demonstrate that simple physical entrapment of magnetic microparticles within the extracellular space of stem cells spheroids during initial formation enables scaffold-free immobilization, translocation and directed assembly of multi-cellular aggregates across multiple length and time scales, even under dynamic suspension culture conditions. The response of aggregates to externally applied magnetic fields was a direct function of microparticle incorporation, allowing for rapid and transient control of the extracellular environment as well as separation of heterogeneous populations. In addition, spatial patterning of heterogeneous spheroid populations as well as individual multi-cellular aggregates was readily achieved by imposing temporary magnetic fields. Overall, this approach provides novel routes to examine stem cell differentiation and tissue morphogenesis with applications that encompass the creation of new model systems for developmental biology, scaffold-free tissue engineering strategies and scalable bioprocessing technologies.