Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis

Abstract

Non-adherent bone marrow-derived cells (NA-BMCs) are a mixed cell population that can give rise to multiple mesenchymal phenotypes and that facilitates hematopoietic recovery. We characterized NA-BMCs by flow cytometry, fibroblast colony-forming units (CFU-f), real-time PCR, and in in vivo experiments. In comparison to adherent cells, NA-BMCs expressed high levels of CD11b(+) and CD90(+) within the CD45(+) cell fraction. CFU-f were significantly declining over the cultivation period, but NA-BMCs were still able to form CFU-f after 5 days. Gene expression analysis of allogeneic NA-BMCs compared to bone marrow (BM) indicates that NA-BMCs contain stromal, mesenchymal, endothelial cells and monocytes, but less osteoid, lymphoid, and erythroid cells, and hematopoietic stem cells. Histopathological data and analysis of weight showed an excellent recovery and organ repair of lethally irradiated mice after NA-BMC transplantation with a normal composition of the BM.

Fig. 1

Comparative flow cytometric analysis of adherent cells and NA-BMCs after 4 days of cultivation (shown for Balb/c). Cells were gated for CD45 expression and co-expression of CD11b, CD90, and CD117 for adherent cells and NA-BMCs

Fig. 2

Colony-forming unit assay (CFU-f) before transplantation. NA-BMCs were cultivated over 5 days and the number of fibroblastic stem cell colonies derived from these cultures was measured (shown for Balb/c and C57Bl/6 mice). Non-adherent BM cells (NA-BMCs) form CFU-fs over the cultivation period. Representative methylene bluestained cultures from mice total BM cells from day 0–4 (colonies from non-adherent supernatant cells in the first cells that became adherent and proliferated) and shown at ×10 of original magnification (a–e)

Fig. 3

Relative expression of CD105, CD117, CD11b, CD133, CD19, CD34, CD38, CD45, CD90, EPCR, GATA-1, GATA-3, Nanog, Osteocalcin, and Sca-1 in NA-BMCs. Gene expression was calculated in relation to expression in bone marrow cells

Fig. 4

Analysis of weight after irradiation, allogeneic transplantation. Transgenic mice (C57Bl/6 background) received allogeneic NA-BMCs (1 × 10⁵–2 × 10⁶), or allogeneic bone marrow cells (2 × 10⁶). Absolute weight (in g) was measured daily

Fig. 5

Flow cytometric analysis of murine CD4, murine MHC-I (H2K^d), murine CD8, human CD4 and HLA-DR3 from day 2–61 after transplantation. MuCD4 (a,b), H-2Kd (c), CD8 (d), huCD4 (e), and HLA-DR (f) levels from peripheral blood (gated for lymphocytes) from days 2–61 after co-transplantation of allogeneic bone marrow and allogeneic splenocytes. Results are shown in comparison to transplantation of syngeneic bone marrow cells

Fig. 6

Distribution of murine CD4 by real-time PCR and immunohistology. Murine CD4 and isotype control immunohistology staining (streptavidine peroxidase technique) and real-time PCR of gut (day 50). Triple transgenic mice (a,b), C57Bl/6 wild-type mice (c,d), allogeneic NA-BMC transplanted mice (e,f). Shown at 20× original magnification. Quantification of murine CD4 by real-time PCR (g)

Fig. 7

Histological analysis by kaolin-aniline-orange G (KAO) staining of knee joints and hematoxylin–eosin (HE) staining for gut and liver from control and transplanted triple transgenic mice (staining day 50). Lethally irradiated mice (a–c), allogeneic NA-BMC transplanted mice (d–f), and allogeneic bone marrow transplanted mice (g–k). Shown at ×20 original magnification