输血是医学的重要组成部分。然而,许多国家都面临着民族的血液危机。为了解决这个持续的血液短缺问题,已经努力在体外产生红细胞(RBC),特别是来自人诱导的多能干细胞(hiPSCs)。然而,用于此目的的hiPSC的最佳来源尚未确定。
在这项研究中,从三种不同的造血干细胞来源-外周血(PB)建立hiPSCs,脐带血(CB)和骨髓(BM)抽吸(每个来源n=3)-使用游离型重编程载体并分化为功能性红细胞。各种时程研究,包括免疫荧光测定,实时定量PCR,流式细胞术,核型分析,形态学分析,氧结合能力分析,进行和RNA测序以检查和比较hiPSC和hiPSC分化的红系细胞的特征。
从三个来源中的每一个建立hiPSC系,并且发现是多能的并且具有相当的特征。所有hiPSCs分化成红系细胞,但是分化和成熟效率存在差异:CB来源的hiPSCs成熟成红系细胞最快,而PB来源的hiPSCs需要更长的成熟时间,但再现性最高.BM来源的hiPSC产生了多种类型的细胞,并表现出较差的分化效率。尽管如此,从所有hiPSC系分化的红系细胞主要表达胎儿和/或胚胎血红蛋白,表明发生了原始红细胞生成。它们的氧平衡曲线全部左移。
集体,PBs和CB来源的hiPSCs都是体外临床生产红细胞的可靠来源,尽管有一些挑战需要克服。然而,由于有限的可用性和生产hiPSC所需的大量CB,以及这项研究的结果,使用PB来源的hiPSCs在体外产生RBC的优势可能超过使用CB来源的hiPSCs的优势。我们相信我们的发现将有助于在不久的将来选择用于体外RBC生产的最佳hiPSC系。
Blood transfusion is an essential part of medicine. However, many countries have been facing a national blood crisis. To address this ongoing blood shortage issue, there have been efforts to generate red blood cells (RBCs) in vitro, especially from human-induced pluripotent stem cells (hiPSCs). However, the best source of hiPSCs for this purpose is yet to be determined.
In this study, hiPSCs were established from three different hematopoietic stem cell sources-peripheral blood (PB), cord blood (CB) and bone marrow (BM) aspirates (n = 3 for each source)-using episomal reprogramming vectors and differentiated into functional RBCs. Various time-course studies including immunofluorescence assay, quantitative real-time PCR, flow cytometry, karyotyping, morphological analysis, oxygen binding capacity analysis, and RNA sequencing were performed to examine and compare the characteristics of hiPSCs and hiPSC-differentiated erythroid cells.
hiPSC lines were established from each of the three sources and were found to be pluripotent and have comparable characteristics. All hiPSCs differentiated into erythroid cells, but there were discrepancies in differentiation and maturation efficiencies: CB-derived hiPSCs matured into erythroid cells the fastest while PB-derived hiPSCs required a longer time for maturation but showed the highest degree of reproducibility. BM-derived hiPSCs gave rise to diverse types of cells and exhibited poor differentiation efficiency. Nonetheless, erythroid cells differentiated from all hiPSC lines mainly expressed fetal and/or embryonic hemoglobin, indicating that primitive erythropoiesis occurred. Their oxygen equilibrium curves were all left-shifted.
Collectively, both PB- and CB-derived hiPSCs were favorably reliable sources for the clinical production of RBCs in vitro, despite several challenges that need to be overcome. However, owing to the limited availability and the large amount of CB required to produce hiPSCs, and the results of this study, the advantages of using PB-derived hiPSCs for RBC production in vitro may outweigh those of using CB-derived hiPSCs. We believe that our findings will facilitate the selection of optimal hiPSC lines for RBC production in vitro in the near future.