Harnessing Mesenchymal Stromal Cells for the Engineering of Human Hematopoietic Niches

Abstract

Tissue engineering opens multiple opportunities in regenerative medicine, drug testing, and modeling of the hematopoiesis in health and disease. Recapitulating the organization of physiological microenvironments supporting leukocyte development is essential to model faithfully the development of immune cells. Hematopoietic organs are shaped by spatially organized niches defined by multiple cellular contributions. A shared feature of immune niches is the presence of mesenchymal stromal cells endowed with unique roles in organizing niche development, maintenance, and function. Here, we review challenges and opportunities in harnessing stromal cells for the engineering of artificial immune niches and hematopoietic organoids recapitulating leukocyte ontogeny both in vitro and in vivo.

Keywords: 3D culture; bone marrow niche; hematopoiesis; mesenchymal stroma cell; organoids model; thymus epithelial cell.

Figure 1

Engineering of murine MSCs to recapitulate human hematopoiesis. MSC from murine bone marrow are isolated and expanded ex-vivo. Those are subsequentially engineered to express human factors and injected together with human HSPCs into the back of an NSG mice in Matrigel plugs. The plug is retreated 2 weeks later. It displays an unorganized structure containing murine MSCs and vasculature together with human hematopoietic progeny. Progeny is dependent on the factors expressed by MSCs.

Figure 2

Different strategies for the generation of humanized ossicles. In vitro expanded human MSCs (from healthy donors or patients) are seeded onto a scaffold, (A) primed to differentiate in cartilage (B) or cultured on Matrigel® (C) Some protocols include the co-seeding of human ECs and the supplement of osteogenic factors, such as BMPs and PTH. Following the first in vitro step, the implantable structures are transplanted subcutaneously into the back of immunodeficient mice for the ossicle formation (D). Aberrant/normal human HSPCs can be added to the system by direct intra-ossicle transplantation (E) or intravenous injection (F) either before or after the implantation of the ossicle. Irradiation is usually performed to promote engraftment. As an alternative, HSPCs can be seeded onto the Matrigel® plug before the in vivo implant (G). The whole process can take several months. The resulting ossicle collected from the mouse is composed of both myelo-supportive marrow stroma and hematopoietic tissues and its progeny of human origin (H). Sinusoidal endothelium, nerve fibers, residual hematopoietic cells, and osteoclasts are derivatives of mouse host.

Figure 3

Different strategies to recapitulate T cell ontogeny using MSCs. 2D co-culture of HPCs (hematopoietic progenitor cells) on bioengineered MSC-derived OP9 cells expressing Notch ligands DLL1/DLL4 with recombinant IL-7 and FLT3-L (A). Isolation of murine thymic lobes from day 14 to 15 old mouse embryos followed by their in vitro culture with deoxyguanosine for 5–7 days to deplete intra-thymic T cells. The thymic lobes are then repopulated by different T cell progenitors and cultured on the surface of membranes as fetal thymic organ culture (FTOC). For reaggregation thymic organ culture (RTOC) thymic stromal cells (thymic SCs) are extracted from the lobes and reaggregated with T cell progenitors by centrifugation. The cell suspension is cultured on a filter membrane in vitro (B). Post-natal human thymus is dissociated in TECs and thymus mesoderm (TM) and further reaggregated with human CD34⁺ cells leading to the formation of thymic organoids. These organoids support the development of mature human T cells when kept in culture or when engrafted within the quadriceps muscle sheath of NSG mice (C). Artificial thymic organoids (ATOs) are generated by the centrifugation of bioengineered MS5 cells expressing Notch ligands DLL1/DLL4 with HSPCs or iPSCs, respectively. After resuspension in a small amount of culture medium the cell suspension is placed on a membrane at the air-liquid interface to form 3D aggregates. This method recapitulates human lymphopoiesis and offers the ability to generate conventionally naïve T cells from HSPCs or iPSCs in vitro (D).