搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication

We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post-mitotic,HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8-10 weeks for the production of functional mature motoneurons. In comparison with other methods,our protocol does not use feeder cells,has a minimum dependence on proteins (purmorphamine replacing SHH),has controllable adherent selection and is adaptable for scalable suspension culture. View Publication

Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines,and we describe their pluripotent phenotype and ability to differentiate into erythroid cells,monocytes,and endothelial cells. More significantly,however,when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells,we observed the formation of pre-B cells. These cells were CD45(+)CD19(+)CD10(+) and were positive for transcripts Pax5,IL7αR,λ-like,and VpreB receptor. Although they were negative for surface IgM and CD5 expression,iPS-derived CD45(+)CD19(+) cells also exhibited multiple genomic D-J(H) rearrangements,which supports a pre-B-cell identity. We therefore have been able to demonstrate,for the first time,that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage. View Publication

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections,and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2),which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs,HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However,HERVK is transcriptionally silenced by the host,with the exception of in certain pathological contexts such as germ-cell tumours,melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations,together with transactivation by OCT4 (also known as POU5F1),synergistically facilitate HERVK expression. Consequently,HERVK is transcribed during normal human embryogenesis,beginning with embryonic genome activation at the eight-cell stage,continuing through the emergence of epiblast cells in preimplantation blastocysts,and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably,we detected HERVK viral-like particles and Gag proteins in human blastocysts,indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product,the HERVK accessory protein Rec,in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection,suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover,Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy,indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development. View Publication

Derivation of functional vascular smooth muscle cells (VSMCs) from human induced pluripotent stem cells (hiPSCs) to generate tissue-engineered blood vessels (TEBVs) holds great potential in treating patients with vascular diseases. Herein,hiPSCs were differentiated into alpha-smooth muscle actin ($$-SMA) and calponin-positive VSMCs,which were seeded onto polymer scaffolds in bioreactors for vascular tissue growth. A functional TEBV with abundant collagenous matrix and sound mechanics resulted,which contained cells largely positive for $$-SMA and smooth muscle myosin heavy chain (SM-MHC). Moreover,when hiPSC-derived TEBV segments were implanted into nude rats as abdominal aorta interposition grafts,they remained unruptured and patent with active vascular remodeling,and showed no evidence of teratoma formation during a 2-week proof-of-principle study. Our studies represent the development of the first implantable TEBVs based on hiPSCs,and pave the way for developing autologous or allogeneic grafts for clinical use in patients with vascular disease. View Publication

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized,the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here,using a short fragment of laminin 411 (LM411-E8),an ECM predominantly expressed in the vascular endothelial basement membrane,we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (textgreater95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate. View Publication

Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets,the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study,we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation,cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating,even after cell sheets were detached from culture dishes. Furthermore,extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue. View Publication

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts,and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer,retaining a pre-inactivation X chromosome state,and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF,naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells,they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression,pronounced tendency for X chromosome inactivation in most female human ES cells,increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells,from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells,and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively,our findings establish new avenues for regenerative medicine,patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo. View Publication

As autologous induced pluripotent stem cell (iPSC) therapy requires a custom-made small-lot cell production line,and the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically,mass culture to produce stock is no longer necessary; instead,a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A three-dimensional (3D) culture method using small,closed-cell manufacturing devices is suitable for autologous iPSC therapy. The use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study,atelocollagen beads were evaluated as a 3D biomaterial to assist 3D culture in the establishment,expansion culture,and induction of differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of a two-dimensional (2D) culture when laminin-511 is added to the medium. In conclusion,atelocollagen beads enable 3D culture of iPSCs,and the quality of the obtained cells is at the same level as those derived from 2D culture. View Publication