RESUMO
Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.
Assuntos
Meios de Cultura/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células-Tronco Pluripotentes , Proteína Desglicase DJ-1/metabolismo , Técnicas de Cultura de Células , Proliferação de Células , Humanos , Fator 4 Semelhante a Kruppel , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
This study aimed to investigate the origin of diverse pathotypes of E. coli, isolated from communal water sources and from the actual drinking water vessel at the point-of-drinking inside households in a low-income urban community in Arichpur, Dhaka, Bangladesh, using a polymerase chain reaction (PCR). Forty-six percent (57/125, CI 95%: 41-58) of the isolates in the point-of-drinking water and 53% (55/103, CI 95%: 45-64) of the isolates in the source water were diarrheagenic E. coli. Among the pathotypes, enterotoxigenic E. coli (ETEC) was the most common, 81% (46/57) of ETEC was found in the point-of-drinking water and 87% (48/55) was found in the communal source water. Phylogenetic group B1, which is predominant in animals, was the most frequently found isolate in both the point-of-drinking water (50%, 91/181) and in the source (50%, 89/180) water. The phylogenetic subgroup B23, usually of human origin, was more common in the point-of-drinking water (65%, 13/20) than in the source water (35%, 7/20). Our findings suggest that non-human mammals and birds played a vital role in fecal contamination for both the source and point-of-drinking water. Addressing human sanitation without a consideration of fecal contamination from livestock sources will not be enough to prevent drinking-water contamination and thus will persist as a greater contributor to diarrheal pathogens.