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

Human embryonic stem cells are derived from the inner cell mass of preimplantation embryo at the blastocyst stage and their differentiation occurs through an intermediate step involving the formation of embryoid bodies (EBs),which are aggregates of embryonic stem cells. The EBs seem to be a powerful tool for investigating the development of embryos,as they can mimic the initial stages of embryonic development. In this study,we aimed to investigate the effect of estrogen compounds on the proliferation and differentiation of short-term and long-term cultured EBs in vitro. For this study,10-day-old (short-term cultured) and 30-day-old (long-term cultured) EBs were subjected to estradiol (E2),estriol (E3),selective estrogen receptor modulator (raloxifene [RLX]),bisphenol A,and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole for 7 days. To confirm the effects of estrogen treatment,ICI-182780 was added to the respective EBs for additional 7 days following estrogen treatment. Quantitative reverse transcription-polymerase chain reaction was performed to analyze the relative expression of differentiation marker genes representing the 3 germ layers. The expression of 7 marker genes,which included α-fetoprotein,hepatocyte nuclear factor (HNF)-3β,HNF-4α (endoderm),brachyury,cardiac actin ([cACT]; mesoderm),nestin (ectoderm),and Oct-4 (undifferentiated),was measured. Significantly,lower expression of HNF-4α in both short-term and long-term cultured EBs was observed after treatment of estrogen compounds compared to control. The expression of HNF-3β in short-term cultured EBs has been positively affected by E2,E3,and RLX. Regarding cACT,higher expression was observed after treatment of E2 (10(-7) mol/L) and E3 (10(-9) mol/L) in short-term cultured EBs,but opposite effects were demonstrated in long-term cultured EBs. The lower expressions of HNF-4α by E2 and RLX were negated by ICI-182780 treatment,although these findings were not statistically significant in E3-treated group. These findings suggest that estrogen compounds have effects on endodermal and mesodermal differentiation of human EBs. View Publication

The in vitro differentiation of human embryonic stem cells (hESCs) offers a model system to explore human development. Humans with mutations in the transcription factor Aristaless Related Homeobox (ARX) often suffer from the syndrome X-linked lissencephaly with ambiguous genitalia (XLAG),affecting many cell types including those of the pancreas. Indeed,XLAG pancreatic islets lack glucagon and pancreatic polypeptide-positive cells but retain somatostatin,insulin,and ghrelin-positive cells. To further examine the role of ARX in human pancreatic endocrine development,we utilized genomic editing in hESCs to generate deletions in ARX. ARX knockout hESCs retained pancreatic differentiation capacity and ARX knockout endocrine cells were biased toward somatostatin-positive cells (94% of endocrine cells) with reduced pancreatic polypeptide (rarely detected),glucagon (90% reduced) and insulin-positive (65% reduced) lineages. ARX knockout somatostatin-positive cells shared expression patterns with human fetal and adult $$-cells. Differentiated ARX knockout cells upregulated PAX4,NKX2.2,ISL1,HHEX,PCSK1,PCSK2 expression while downregulating PAX6 and IRX2. Re-expression of ARX in ARX knockout pancreatic progenitors reduced HHEX and increased PAX6 and insulin expression following differentiation. Taken together these data suggest that ARX plays a key role in pancreatic endocrine fate specification of pancreatic polypeptide,somatostatin,glucagon and insulin positive cells from hESCs. View Publication

An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry,expansion of the progenitor population,and delayed neuronal differentiation,all key features of the developing human cortex. In contrast,Pten deletion in mouse organoids does not lead to folding. Finally,we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex. View Publication

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment,tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors,whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms),suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However,CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically,the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer,tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly,in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion,the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress. View Publication