Systematic optimization of an engineered hydrogel allows for selective control of human neural stem cell survival and differentiation after transplantation in the stroke brain

Abstract

Stem cell therapies have shown promise in promoting recovery in stroke but have been limited by poor cell survival and differentiation. We have developed a hyaluronic acid (HA)-based self-polymerizing hydrogel that serves as a platform for adhesion of structural motifs and a depot release for growth factors to promote transplant stem cell survival and differentiation. We took an iterative approach in optimizing the complex combination of mechanical, biochemical and biological properties of an HA cell scaffold. First, we optimized stiffness for a minimal reaction of adjacent brain to the transplant. Next hydrogel crosslinkers sensitive to matrix metalloproteinases (MMP) were incorporated as they promoted vascularization. Finally, candidate adhesion motifs and growth factors were systemically changed in vitro using a design of experiment approach to optimize stem cell survival or proliferation. The optimized HA hydrogel, tested in vivo, promoted survival of encapsulated human neural progenitor cells (iPS-NPCs) after transplantation into the stroke core and differentially tuned transplanted cell fate through the promotion of glial, neuronal or immature/progenitor states. This HA hydrogel can be tracked in vivo with MRI. A hydrogel can serve as a therapeutic adjunct in a stem cell therapy through selective control of stem cell survival and differentiation in vivo.

Keywords: Angiogenesis; Astrocyte; Hyaluronan; Neural stem cell; Regeneration; iPS.

Figure 1. Mechanical and composition gel optimization

(A) Schematic illustration of the injectable hyaluronic acid (HA) composed of acrylated hyaluronic acid, MMP degradable or non-degradable motifs, adhesion peptides and heparin bound growth factors. (B) Characterization of stiffness and crosslinker. (Bi) Schematic illustration of brain coronal sections and their orientations. The stroke cavity is represented by the asterisk. (Bii, Biii) TTC-stained brain sections showing infarcted tissue (delimitated by dotted line) without (Bii) and with (Biii) injection of a 350 Pa gel in the stroke cavity. (Biv, Bv) Vascular laminin staining (green) in HA gel (red) at 2 weeks post-injection with an MMP insensitive (or MMP-In; Biv) or sensitive (or MMP-Sen; Bv) cross-linker. (Bvi) Quantification of gel volume, (Bvii) infarcted tissue and (Bviii) the vascular density in the injected HA gels. DOE approach to optimize concentrations of (C) soluble or (D) bound growth factors. iPS-NPCs survival in HA gel at 1 week in response to varying concentrations of (Ci) soluble or (Di) bound growth factors. DOE prediction for the maximized and minimized cell proliferation for (Cii) soluble and (Dii) bound growth factors. (E) Combining optimal growth factor concentrations. (Ei) iPS-NPCs survival, (Eii) sprouting (Dcx staining) and (Eiii, Eiv) differentiation (% of Dcx cells by flow cytometry) in the combined optimization HA Max and HA Min at 1 week. (F) Schematics of the final optimized condition for in vivo testing and table of the gel component concentration. HA Nopt demonstrates non-optimized gel with equimolar concentrations of adhesion motifs and no GFs. Scale bars represent 1 mm (Bii, Biii), 100 μm (Biv, Bv) and 50 μm (Eiv). *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively.

Figure 2. Biomaterial infiltration and resorption in vivo

Fluorescent image of stroke area (A) 2 weeks and (B) 6 weeks after transplantation with 100,000 iPS-NPCs (green) encapsulated in HA Max (blue), or (C) 6 weeks after transplantation of 50,000 iPS-NPCs encapsulated in HA Max. (D) Brain MRI images of animals at 9 weeks after stroke (dashed rectangle), (E) stroke plus HA Max injection and (F) stroke plus transplantation of 100,000 cells encapsulated in HA Max. (J) Quantification of gel representing T2 signal (arrowhead in E and F). (K) Fluorescent staining of vascular growth within HA Max at two weeks after transplantation, where the area demarcated in dashed line is magnified in L (merged), M (transplanted cells, in green), N (CD31 positive endothelial cells, in red) and O (gel, in blue). Scale bar represents 100 μm (A–C) and 10 μm (K–H). ** indicates P < 0.01.

Figure 3. Cell proliferation, differentiation and survival in HA gel

(A) Fluorescent image and (B) analyses of brain sections injected with 100,000 cells stained at 2 weeks for the human-specific nuclear antigen HuNu and the proliferation marker Ki67, and at 6 weeks for HuNu, GFP, the neural precursor marker SOX2 and the neuronal antigen NF200. (C) Survival of transplanted cells with and without prior encapsulation was analyzed at 2 weeks post-injection on brain section stained for GFP. Ratio of GFP positive area was quantified in each group. Scale bars represents 25 μm (A) and 200 μm (C). * indicates P < 0.05.

Figure 4. Effect of HA Max composition on astrocytic cell differentiation

(A) Fluorescent images and (B) analyses of brain sections injected with 100,000 cells stained at 6 weeks for GFP, GFAP, as the marker for both astrocytes and NPCs, and S100b that is more specific to astrocytes. The numbers of GFP/GFAP as well as GFP/GFAP/S100b positive cells were normalized to the total GFP cells to obtain a percentage cells expressing one or both astrocytic markers. In addition, triple labeled cells were normalized to the number of GFP-GFAP cells in order to estimate the percentage of GFAP cells that are also positive for the more exclusive astrocytic marker, S100b. Scale bars, 50 μm. **** indicates P < 0.0001.