Bhardwaj Rashmi, Kumar Lalit, Chhabra Deepika, Mehra N K, Sharma Atul, Mohanty Sujata, Kochupillai Vinod
Institute Rotary Cancer Hospital (IRCH), All India Institute of Medical Sciences (AIIMS), New Delhi, India.
Sri Sri Institute For Advanced Research (SSIAR), Ved Vignan Maha Vidhya Peeth (VVMVP), F003 Soudhamini Apartment, 21st Kanakpura Road, Art of Living International Center, Udaipura, Bengaluru, 560082, India.
Sci Rep. 2021 Jun 4;11(1):11879. doi: 10.1038/s41598-021-91272-6.
Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34 fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34 & CD34) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.
胎儿肝脏造血干细胞（HSPCs）因其增殖潜力，一直被认为适合用于再生障碍性贫血的治疗。在某些情况下，骨髓恢复是可能的；然而，这些细胞的植入潜力并不理想，这可能是由于从单个胎儿中获取的此类细胞数量较少。本研究探索了如何在体外条件下扩增胎儿肝脏造血干细胞。我们从胎儿肝脏中分离出单核细胞，并通过细胞表面标志物CD34对造血干细胞进行鉴定和分析。通过磁性细胞分选阳性选择法分离出CD34胎儿肝脏造血干细胞。造血干细胞（CD34）在无血清培养基中，使用5种细胞因子，即干细胞因子（SCF）、粒细胞巨噬细胞集落刺激因子（GM-CSF）、白细胞介素-6（IL-6）、Fms相关酪氨酸激酶3（FLT-3）和促红细胞生成素（EPO），以4种不同组合并添加补充剂进行培养21天。在整个21天的培养过程中，细胞活力持续大于90%。细胞在培养基、补充剂和5种细胞因子（即SCF、FLT-3、IL-6、EPO和GM-CSF）的组合中扩增效果最佳，可产生大量的总（CD34⁺和CD34⁻）细胞。尽管培养中有核细胞总数显著增加，但在此期间CD34抗原表达水平却稳步下降。
