The Hematopoietic Reconstitution through Intra-Bone-Marrow Injection Using Murine Fetal Liver Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) were considered an effective treatment option for many blood diseases and autoimmune diseases. HSCs can currently be isolated from bone marrow and umbilical cord blood. Due to the shortage of these resources, an alternative source of HSCs preparation were sorted out in this study and the ability of hematopoietic reconstruction were examined using such HSCs obtained from fetal liver.

Fetal liver hematopoietic stem cells (FL-HSCs) were isolated from embryos of transgenic mice with an erythoid-specific GFP expression that was driven by a human β-globin gene promoter (HS23-GFP mice). Thus, GFP expression could be used as tracer of marker genes of red blood cells derived from the donor FL-HSCs visually. Single-cell suspension of E13.5 HS23-GFP murine liver was used for Ficoll density gradient separation, and the FL-HSC enriched portion was collected for intra-bone-marrow injection (IBMI) into 6-8 week-old recipient mice post 137Cs γ irradiation treatment. Three doses were tested for injection, i.e. 1×10⁵, 1×10⁶ and 1×10⁷ cells, respectively.

Hematopoietic reconstruction ability was observed every week after injection using blood smear and routine blood panel such as RBC, HGB and HCT. Bone marrow smear and pathological sections of liver and spleen of recipient mice were analyzed 8 weeks post FL-HSC transplantation. GFP expressed erythrocyte was captured in the peripheral blood starting one week after HL-HSC transplantation. The ability of hematopoietic reconstruction continued to rise until week 7, and became stable thereafter. Among the three treatment groups, the highest cell number group (1×10⁷ ) had the best hematopoietic restoration, according to the RBC, HGB and HCT data. No abnormality such as lymphocytic infiltration in pathological sections of liver and spleen of recipients were noted. Bone marrow smear of recipient mice showed no difference when comparing to that of the wide type mice. This demonstrated HSC obtained from fetal liver, are capable of reconstructing hematopoietic function after transplantation using IBMI. This could provide an alternative source of HSC for future study of hematopoietic function and for future therapeutic treatment for blood diseases.