An in vivo model to study and manipulate the hematopoietic stem cell niche

Abstract

Because the microenvironment that supports hematopoietic stem cell (HSC) proliferation and differentiation is not fully understood, we adapted a heterotopic bone formation model as a new approach for studying the HSC microenvironment in vivo. Endogenous HSCs homed to tissue-engineered ossicles and individually sorted HSCs from ossicles were able to reconstitute lethally irradiated mice. To further explore this model as a system to study the stem cell niche, ossicles were established with or without anabolic parathyroid hormone (PTH) treatment during the 4-week course of bone development. Histology and micro-computed tomography showed higher bone area-to-total area ratios, thicker cortical bone and trabecular bone, significantly higher bone mineral density and bone volume fraction in PTH-treated groups than in controls. By an in vivo competitive long-term reconstitution assay, HSC frequency in the ossicle marrow was 3 times greater in PTH groups than in controls. When whole bone marrow cells were directly injected into the ossicles after lethal irradiation, the PTH-treated groups showed an enhanced reconstitution rate compared with controls. These findings suggest the residence of HSCs in heterotopic bone marrow and support the future use of this ossicle model in elucidating the composition and regulation of the HSC niche.

Figure 1

Low- and high-magnification images of H&E-stained ossicle sections and micro-CT images (inset image). PTH-treated ossicles contained a higher density of trabecular bone and bone marrow cells. (A) Ossicle in control group without PTH treatment (×40). (B) Ossicle in PTH-treated group (×40). (C) Ossicle in control group (×100). (D) Ossicle in PTH-treated group (×100).

Figure 2

Flow cytometric analysis of peripheral blood and bone marrow of ossicles shows the origin of the ossicle bone marrow. Ossicles were made subcutaneously in CD45.2 mice with BMSCs harvested from CD45.1 mice. No CD45.1 donor type hematopoietic cells were found in either the peripheral blood or ossicle bone marrows during the 16 weeks after the implantation period (n = 5). The chart is a representative analysis of ossicle bone marrow.

Figure 3

HSC frequency in ossicle and femur bone marrow measured by in vivo long-term competitive reconstitution assays (n = 3). Bone marrow from PTH-treated ossicles had nearly a 3-fold higher stem cell frequency than the control group (P < .01). The femur bone marrow in the PTH-treated group also displayed a slightly higher of stem cell frequency than the control group. *P < .01. Error bars represent SD.

Figure 4

Long-term reconstitution through direct ossicle injection or retro-orbital injection of HSCs. (A) Direct injection of whole bone marrow into ossicles resulted in long-term reconstitution in 5 of 25 irradiated mice. Each line represents the frequency of donor-derived myeloid, B, or T cells in a single mouse at 4, 8, 12, and 16 weeks after transplantation. The red lines represent mice that were long-term multilineage reconstituted, blue lines represent nonreconstituted mice. (B) Directly injecting whole bone marrow into PTH-treated ossicles reconstituted 10 of 20 irradiated mice.

Figure 5

Long constitution capacity of purified HSCs from ossicle bone marrow. HSCs were identified in ossicle bone marrow by immunofluorescent staining with a combination of CD150, CD48, CD41, MECA32 antibodies. The white arrow depicts a HSC that is CD150⁺ (red; A) but negative for CD48, CD41, Lin (green; B), and MECA32 (pink; C). The nuclei are stained with DAPI (blue). (D) HSCs from ossicles were isolated from the combined gate of CD150⁺CD41⁻CD48⁻ and were confirmed to be positive for Sca-1 and c-kit. (E) Five HSCs harvested from ossicles supported long-term multilineage reconstitution in 2 of 4 lethally irradiated mice. Each line represents the frequency of donor-derived myeloid, B, or T cells in a single mouse at 4, 8, 12, and 16 weeks after transplantation. The black line at 0.3% represents the background threshold, meaning that reconstitution is not considered in mice falling below this line.