Implantable microenvironments to attract hematopoietic stem/cancer cells

Abstract

The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.

Fig. 1.

Biomimetic design of 3D microfabricated scaffolds and human BMSC culture. (A) Scanning electron microscopic images of demineralized bovine cancellous bone at different magnifications. (B) Fabrication scheme with scanning electron microscopy and camera images at corresponding stages. (C) Scanning electron microscopic and reconstructed 3D confocal images of human BMSC coatings in the scaffold. (D) Representation of the local chemical environment created by BMSCs. (E) Efficiency of cell seeding is dependent on cavity size. (F) Normalized IFN-γ secretion of human peripheral blood mononuclear cells with BMSC-conditioned medium and lipopolysaccharide relative to the highest overall value. (G) Comparison of secretion of specific soluble factors by BMSC cultures on a 2D plate and 3D scaffold (D = 150–300 µm). *P < 0.05.

Fig. 2.

Development of vascularized, extramedullary marrow tissue. (A) Gross histological observations 4 wk after implantation show increased vascularization in BMSC-seeded scaffolds. (B) Optical image of corrosion-casted and decellularized BMSC-seeded scaffold. (C) Immunostaining of mCD31⁺ endothelial cells. (D) Quantitative comparison of mCD31⁺ area 4 wk after implantation showing significant increase by BMSCs. (E) Immunohistostaining of mVEGFR3 in BMSC-seeded scaffolds. (F) Dorsal window chamber grafted on top of the implanted scaffold. (G) Intravital imaging of interscaffold vasculature (green, blood vessels; blue, autofluorescent for contrast). (H) Distribution of interscaffold vessel diameters. (I) Histological comparison of H&E-stained tissue sections from unseeded and BMSC-seeded scaffolds. (J) Interscaffold tissue compartment model.

Fig. 3.

Local and systemic hematopoietic cell analysis 4 wk after scaffold implantation. (A) Detection of LSK cells by flow cytometry from explanted scaffolds and endogenous bone marrow cells (n = 4) and (B) their frequency (*P < 0.05). BM, bone marrow. (C) Comparison of linage cell analyses in the bone marrow and scaffold cells. (D) Normalized complete peripheral blood count of scaffold-implanted mice with respect to normal mice.

Fig. 4.

Retention and engraftment of directly transplanted human bone marrow CD34⁺ hematopoietic progenitor cells. (A) Schematic of experimental design of human CD34+ cell transplantation s.c., i.v., or via implant in sublethally irradiated mice. (B) FMT images of 5 × 10⁵ directly injected human CD34⁺ cells prestained with a near-infrared dye over 3 d show longer retention of HSPCs in BMSC-seeded scaffolds. (C) Analysis of human CD45⁺ cells from endogenous bone marrow and implanted scaffolds 16 wk after transplantation of human CD34⁺ cells (n = 4). Two independent trials were performed. (Inset) Immunohistostaining of human CD45⁺ cells in BMSC-seeded scaffolds.

Fig. 5.

Homing of i.v. transplanted human bone marrow cells. (A) Schematic of experimental design. (B) Immunostaining of human nuclei in unseeded and BMSC-seeded scaffolds 3 d after i.v. injection. (C) Confocal images of explanted scaffolds 3 d after i.v. injection of prestained TF-1a cells. (D) Cytofluorimetry of prestained TF-1a cells from the bone marrow and explanted scaffolds. (E and F) Intravital confocal image of (E) unseeded and (F) BMSC-seeded scaffolds 5 h after i.v. injection. (G) Correlation between blood vessel diameters and number of adhered leukemic cells.