Long-term ex vivo expansion of mouse hematopoietic stem cells

Abstract

Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.

Figure 1:. Schematic overview of the HSC expansion culture protocol

HSCs are first isolated from mouse bone marrow (steps 1–18) to initiated to initiate either bulk HSC cultures (step 19A) or clonal HSC cultures (step 19B). Following ex vivo HSC expansion, the derived cultures can then be analyzed in vitro (steps 20–24), or in vivo by transplantation into nonconditioned recipient mice (step 25A) or radiation-conditioned recipient mice (step 25B).

Figure 2:. Representative gating scheme for FACS purification of bone marrow HSCs

Live CD150⁺CD34^-Kit⁺Sca1⁺Lineage^- (CD150⁺CD34^-KSL) HSCs should be isolated by FACS from c-Kit-enriched mouse bone marrow using the following gating scheme, and directly sorted into wells containing fresh HSC media.

Figure 3:. Representative images of mouse HSC cultures

(A) Representative images of usual cell density of bulk HSC cultures after 8 and 10 days. Cultures tend from one side of the well and expand out. The majority of cells should be small and round, although some larger (megakaryocyte-like) cells are expected in the cultures. Where possible, perform media changes from the opposite side of the well to minimize disturbing the cells. Images at 40x magnification. (B) Representative images of expected cell morphology at day 24 and 37 (above) and images displaying evidence of cell differentiation (below). Evidence of cell clumping and cell death are indicators of bad HSC cultures. Images at 40x magnification, with insert images at 200x magnification.

Figure 4:. Representative gating scheme for flow cytometric analysis of HSC cultures

HSC cultures should be analyzed by flow cytometry using the following gating scheme, to determine expression of Kit, Sca1, Lineage, CD150, and PI staining. This example staining profile phenotype is representative of the cultures that should be observed from bulk HSC culture.

Figure 5:. Example flow cytometric analysis of HSC cultures

Example flow cytometry plots of good and bad HSC cultures analyzed at day 28 using the gating scheme described in Figure 4.

Figure 6:. Representative gating scheme for flow cytometric peripheral blood analysis

The peripheral blood of transplantation recipient mice should be analyzed by flow cytometry using the following gating scheme, to determine the relative donor chimerism of engrafting HSCs.

Figure 7:. Representative gating scheme for flow cytometric bone marrow analysis

The bone marrow of transplantation recipient mice should be analyzed by flow cytometry using the following gating scheme, to determine the relative donor chimerism of engrafting HSCs within the CD34^-KSL HSC compartment.