ABSTRACT
【Objective】 Utilizing a specially engineered miR-144-GFP-H1 human embryonic stem cell (hESC) reporter line, this study leverages GFP fluorescence as an indicator of miR-144 expression to gauge the progression of erythropoiesis. The investigation is aimed at elucidating the potential roles of lncRNAs within the erythropoietic framework and conducting an initial assessment of their functional impact. 【Methods】 The miR-144/451-GFP-H1 cell line (hereafter referred to as 144-H1) was utilized for in vitro erythrocyte induction culture. The subpopulations of cells entering the erythropoiesis stage were characterized by the surface molecules CD71 and GPA. The GFP reporter gene of miR-144 served as a critical determinant to distinguish between GFP-positive cells (with a high propensity for erythropoiesis) and GFP-negative cells (with a low propensity for erythropoiesis). Transcriptome sequencing was performed on both groups to identify differentially expressed long non-coding RNAs (lncRNAs). LncRNA entries with potential for validation were selected for preliminary functional verification. The CRISPR/Cas9 gene editing technique was employed to design functional interference strategies for the targeted lncRNAs, obtaining 144-H1 cell lines with knocked-out function of the specific lncRNAs. These knockout cell lines, along with non-knockout 144-H1 cell lines, were used for parallel erythrocyte induction culture to identify differential nodes. This approach preliminarily verified their impact on erythropoiesis in an in vitro development model. 【Results】 1)The constructed 144-H1 cell line was capable of expressing GFP fluorescence upon entering the stage of in vitro erythrocyte induction, indicating the activation of miR-144/451. 2)Within the CD71, GPA double-positive group, significant differences in lncRNA expression were observed between the GFP-positive and GFP-negative subpopulations. 3) Gene editing strategies involving the deletion of sequence segments capable of effectively interfering with the function of multiple lncRNA entries were designed and verified for successful editing. In the knockout cell lines, parts of the lncRNA sequences were directly deleted. 4)In parallel validation experiments of erythrocyte induction culture, cell lines with LINC01569 knocked out exhibited significant differences in flow cytometric subpopulations and cell proliferation capabilities compared to the non-knockout cell lines: ①The knockout cell lines showed sustained high expression of GFP fluorescence. ②The proportion of the CD71-GPA double-positive group in the knockout cell lines continuously decreased during erythrocyte maturation. ③No significant expression of hemoglobin was observed in the knockout cell lines, lacking the characteristic red color. ④The cell proliferation capability of the knockout cell lines was significantly lower than that of the non-knockout cell lines (P<0.05). 【Conclusion】 The successful employment of the 144-H1 cell line facilitated an exploration into the potential functions of lncRNAs in erythropoiesis. This enables the design of more refined in vitro developmental experiments to enhance the precision in capturing lncRNA functions. Among the differentially expressed lncRNA entries, LINC01569 was preliminarily validated to play a regulatory role in erythropoiesis. The functional absence of LINC01569 severely impacts the normal differentiation and proliferation of erythrocytes. The specific regulatory mechanism of LINC01569 in erythropoiesis warrants further investigation and research.
ABSTRACT
【Objective】 To explore the establishment methods of transgenic human umbilical cord mesenchymal stem cells (hUC-MSCs) overexpressing tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) based on the transposons, and attempt to apply it on the nude mice mode with glioma. 【Methods】 PiggyBac transposon system specially designed by us was used to prepare non-targeting and Her2-targeting hUC-MSCs that can stably express TRAIL through puromycin screening. The glioma cells expressing firefly luciferase (U87MG-LUC) were injected into the skull of the immunodeficient mice (BALB/c-nu/nu) with 1×106 cells per mouse. After 7 days of injection, the mice transplanted with U87MG were detected with a small animal living imager to determine the size and location of the tumors in skull. Then we injected the glioma-transplantation nude mouse with two kinds of transgenic hUC-MSCs expressing TRAIL (named as untarget-TRAIL and target-TRAIL, respectively), or the non-transgenic hUC-MSCs (all 1×106 cells per mouse) or PBS (named as WT-MSCs and PBS for negative control) respectively, and then monitored the changes of tumor signals by a small animal living imager every week for 3~4 weeks. 【Results】 After six passages to expand the cells, the both transgenic cell lines can stably express TRAIL gene. Their ratio of green fluorescent protein (GFP) positive cells can reach 93%-97%, and the positive ratio of their MSC-specific surface markers still maintained normal (CD34+, CD45+, and HLA-DR+ all <0.1%, CD90>99%, CD73>88%, and CD105 >60%). The median survival time (d) of U87MG-transplanted nude mice in the groups of untarget-TRAIL, target-TRAIL, WT-MSCs, and PBS was 41 vs 39 vs 24 vs 23(P<0.05). 【Conclusion】 The transgenic hUC-MSCs overexpressing TRAIL gene can significantly prolong the survival time of nude mice with brain glioma.
ABSTRACT
Background and Objectives@#p21, an important member of the Cip/Kip family, is involved in inhibitory effects of RUNX1b overexpression during the early stage of human hematopoiesis. @*Methods@#and Results: We established a human embryonic stem cell (hESC) line with inducible expression of p21 (p21/hESCs). Overexpression of p21 did not influence either mesoderm induction or emergence of CD34＋ cells, but it significantly decreased the production of CD43＋ cells and changed the expression profile of hematopoiesis-related factors, leading to the negative effects of p21 on hematopoiesis. @*Conclusions@#In RUNX1b/hESC co-cultures when RUNX1b was induced from D0, perturbation of the cell cycle caused by upregulation of p21 probably prevented the appearance of CD43＋ cells, but not CD34＋ cells. The mechanisms via which CD34＋ cells are blocked by RUNX1b overexpression remain to be elucidated.