Abstract
BackgroundHematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases. However, limited number of functional HSCs hinders successful transplantation. The purpose of our current study is to develop a new and cost-efficient medium formulation that could greatly enhance the expansion of HSCs while retaining their long-term repopulation and hematopoietic properties for effective clinical transplantation.MethodsEnriched human CD34+ cells and mobilized nonhuman primate peripheral blood CD34+ cells were expanded with a new, cost-efficient expansion medium formulation, named hematopoietic expansion medium (HEM), consisting of various cytokines and nutritional supplements. The long-term repopulation potential and hematologic-lineage differentiation ability of expanded human cells were studied in the non-obese diabetic/severe combined immunodeficiency mouse model. Furthermore, the efficacy and safety studies were performed by autologous transplantation of expanded primate cells in the nonhuman primate model.ResultsHEM could effectively expand human CD34+ cells by up to 129 fold within 9 days. Expanded HSCs retained long-term repopulation potential and hematologic-lineage differentiation ability, as indicated by (1) maintenance (over unexpanded HSCs) of immunophenotypes of CD38−CD90+CD45RA−CD49f+ in CD34+ cells after expansion; (2) significant presence of multiple human hematopoietic lineages in mouse peripheral blood and bone marrow following primary transplantation; (3) enrichment (over unexpanded HSCs) in SCID-repopulating cell frequency measured by limiting dilution analysis; and (4) preservation of both myeloid and lymphoid potential among human leukocytes from mouse bone marrow in week 24 after primary transplantation or secondary transplantation. Moreover, the results of autologous transplantation in nonhuman primates demonstrated that HEM-expanded CD34+ cells could enhance hematological recovery after myelo-suppression. All primates transplanted with the expanded autologous CD34+ cells survived for over 18 months without any noticeable abnormalities.ConclusionsTogether, these findings demonstrate promising potential for the utility of HEM to improve expansion of HSCs for clinical application.
Highlights
Hematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases
Growth factor optimization for human HSC expansion Various growth factors reagents, including TPO, IL-3, granulocyte colonystimulating factor (G-CSF), GM-CSF, and StemRegenin 1 (SR1), were added one by one, or in combination, into the media Iscove’s modified Dulbecco’s medium (IMDM) supplemented with nutrition supplements, as well as stem cell factor (SCF) and Fms-related tyrosine kinase 3 ligand (Flt-3 L), to identify the optimal concentrations needed for expanding human HSCs ex vivo (Table 1)
The results show that mice transplanted with unexpanded HSCs and a low dose of expanded HSCs have similar proportions of engraftment, and mice infused with a high dose of expanded HSCs contain even more human cells, indicating that hematopoietic expansion medium (HEM)-expanded human HSCs retain bone marrow (BM) engraftment and long-term repopulation ability in vivo
Summary
Hematopoietic stem cell (HSC) transplantation has been widely applied to the treatment of malignant blood diseases. The purpose of our current study is to develop a new and cost-efficient medium formulation that could greatly enhance the expansion of HSCs while retaining their long-term repopulation and hematopoietic properties for effective clinical transplantation. Methods: Enriched human CD34+ cells and mobilized nonhuman primate peripheral blood CD34+ cells were expanded with a new, cost-efficient expansion medium formulation, named hematopoietic expansion medium (HEM), consisting of various cytokines and nutritional supplements. Reasonable stem cell expansion has been achieved by coculturing with BM mesenchymal stromal cells, with immortalized stromal cells [12,13,14] or by over-expression of self-renewal genes such as HOXB4 [15,16,17] and Sall4b [18, 19] These approaches involve the manipulation of the stromal environment or perturbation of the HSC genome, at the risk of unintended adverse effects, such as carcinogenesis. There is still a large need to further optimize efficiency and decrease cost
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