Three-dimensional co-culture of mesenchymal stromal cells and differentiated osteoblasts on human bio-derived bone scaffolds supports active multi-lineage hematopoiesis in vitro: Functional implication of the biomimetic HSC niche

Abstract

Recent studies have indicated that the hematopoietic stem/progenitor cell (HSPC) niche, consisting of two major crucial components, namely osteoblasts (OBs) and mesenchymal stromal cells (MSCs), is responsible for the fate of HSPCs. Thus, closely mimicking the HSPC niche ex vivo may be an efficient strategy with which to develop new culture strategies to specifically regulate the balance between HSPC self-renewal and proliferation. The aim of this study was to establish a novel HSPC three-dimensional culture system by co-culturing bone marrow-derived MSCs and OBs differentiated from MSCs without any cytokines as feeder cells and applying bio-derived bone from human femoral metaphyseal portion as the scaffold. Scanning electron microscopy revealed the excellent biocompatibility of bio-derived bone with bone marrow-derived MSCs and OBs differentiated from MSCs. Western blot analysis revealed that many cytokines, which play key roles in HSPC regulation, were comprehensively secreted, while ELISA revealed that extracellular matrix molecules were also highly expressed. Hoechst 33342/propidium iodide fluorescence staining proved that our system could be used to supply a long-term culture of HSPCs. Flow cytometric analysis and qPCR of p21 expression demonstrated that our system significantly promoted the self-renewal and ex vivo expansion of HSPCs. Colony-forming unit (CFU) and long-term culture-initiating cell (LTC-IC) assays confirmed that our system has the ability for both the expansion of CD34+ hematopoietic stem cells (HPCs) and the maintenance of a primitive cell subpopulation of HSCs. The severe-combined immunodeficient mouse repopulating cell assay revealed the promoting effects of our system on the expansion of long-term primitive transplantable HSCs. In conclusion, our system may be a more comprehensive and balanced system which not only promotes the self-renewal and ex vivo expansion of HSPCs, but also maintains primitive HPCs with superior phenotypic and functional attributes.

Figure 1

Osteoblasts differentiated from human marrow mesenchymal stromal cells (MSCs). (A) Morphology of HSPCs in the 2D system. The mixture of MSCs and osteoblasts (OBs) induced from the bone marrow-derived MSCs (BMSCs) as seed cells was seeded in a 24-well plate to build the 2D system in which the HSPCs were cultured for 2 weeks (×200 magnification). (B) Morphology of human MSCs of the first generation (×200 magnification); (C) morphology of osteoblasts differentiated from MSCs after 10 days (×40 magnification); (D) Gomori staining for alkaline phosphatase (ALP) expression in osteoblasts after 8 days (×200 magnification); (E) assay for osteocalcin expression with rhodamine-conjugated monoclonal antibody after 14 days (×400 magnification); (F) assay for collagen I by immunohistochemistry after 10 days (×100 magnification); (G) Alizarin S staining for mineralized nodule formation after 21 days (×100 magnification).

Figure 2

The cells grew after seeding human bone marrow stromal cells (BMSCs) and osteoblasts differentiated from mesenchymal stromal cells (MSCs) onto bio-derived bone for 7–10 days. (A and B) Human BMSCs and osteoblasts differentiated from MSCs on human bio-derived bone scaffolds examined under an inverted microscope after 10 days [(A) ×100 magnification; (B) ×200 magnification]; (C and D) human BMSCs and osteoblasts differentiated from MSCs on human bio-derived bone scaffolds examined under a scanning electron microscope after 10 days [(C and D) ×1,000 magnification; arrows indicate ECM components]; (E and F) Hoechst 33342/propidium iodide (PI) fluorescent staining to examine the frequency of live and dead feeder cells on trabecular bone of bio-derived bone scaffolds after 2 weeks of HSPC culture. (E) Observation under an inverted microscope; (F) observation under a fluorescence microscope. Blue indicates the live cells and red indicates the dead cells.

Figure 3

3D-Mix of mesenchymal stromal cells (MSCs) and osteoblasts differentiated from MSCs supply a closely similar microenvironment. (A) Western blot analysis revealed the expression of angiopoietin-1, RUNX2, CXCL12, osteopontin and stem cell factor (SCF) in the 4 culture systems. (B–E) ELISA confirmed the expression of extracellular matrix proteins, such as fibronectin, collagen IV, vitronectin and laminin in the 4 culture systems. ^*p<0.05 compared with 3D-Mix system.

Figure 4

CD34⁺ cells in the 4 culture systems. (A) Flow cytometric analysis was used to count the number of CD34⁺ cells, CD34⁺CD38⁻ cells and CD34⁺CD38⁺ cells at 14 days; (B) qPCR assay was used to determine the expression of p21; (C) colony formation assays; (D) CD34⁺ cells generated in the 4 culture systems were subjected to in vitro long term culture initiating cell (LTC-IC) assays to determine whether they could preserve the ability to sustain long-term hematopoiesis. ^*p<0.05 compared with 3D-Mix system.

Figure 5

Determination of human hematopoietic reconstitution in NOD/SCID mice 8 weeks post-transplantation. (A) Transplantated bone marrow cells belonging to the 3D-Mix system from the mice were subjected to flow cytometric analysis; (B) PCR analysis of 17α-satellite gene expression demonstrated the presence of human hematopoietic cells in the bone marrow of NOD/SCID mice.