In vitro expansion of fetal liver hematopoietic stem cells

N K Mehra,Deepika Chhabra,Rashmi Bhardwaj,Sujata Mohanty,Vinod Kochupillai,Atul Sharma,Lalit Kumar

doi:10.1038/s41598-021-91272-6

Abstract

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.

Highlights

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential
HSPCs have been used to treat a number of nonmalignant conditions such as Aplastic Anemia (AA), Severe Combined Immunodeficiency Disease (SCID), Congenital Metabolic Disorders, and malignant conditions such as Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Myelodysplastic Syndrome (MDS), Chronic Myeloid Leukemia (CML), and Multiple Myeloma (MM)
Fetal liver mononuclear cells (MNC) through flowcytometry showed that the total C D34+ HSPCs population ranged from 1.2% to 12.8% with a median value of 5.5%

Summary

Introduction

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. 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. The proportion of HSPCs (marked by surface antigen CD 34) in the fetal liver are greater than those found in cord blood and bone marrow; and are more or less equal to those found in mobilized peripheral b lood[5]. Fetal liver hematopoietic stem and progenitor cells (FL-HSPCs) have been found to exhibit greater proliferative potential compared to the adult bone marrow and cord blood. Expanded fetal liver CD34+ HSPCs produced under ‘‘in vitro’’ conditions may provide large homogenous population of cells from a single fetal liver source

Methods

Results

Discussion

Conclusion