Enabling stem cell therapies for tissue repair: current and future challenges

Abstract

Stem cells embody the tremendous potential of the human body to develop, grow, and repair throughout life. Understanding the biologic mechanisms that underlie stem cell-mediated tissue regeneration is key to harnessing this potential. Recent advances in molecular biology, genetic engineering, and material science have broadened our understanding of stem cells and helped bring them closer to widespread clinical application. Specifically, innovative approaches to optimize how stem cells are identified, isolated, grown, and utilized will help translate these advances into effective clinical therapies. Although there is growing interest in stem cells worldwide, this enthusiasm must be tempered by the fact that these treatments remain for the most part clinically unproven. Future challenges include refining the therapeutic manipulation of stem cells, validating these technologies in randomized clinical trials, and regulating the global expansion of regenerative stem cell therapies.

Figure 1. Schematic of microfluidic chip-based single cell transcriptional analysis

Single cells are sorted by fluorescence-activated cell sorting into individual wells of a 96-well plate that has been pre-loaded with reverse transcriptase polymerase chain reaction reagents. cDNA is created for each gene target within each individual cell followed by an initial pre-amplification step. Single cell cDNA and primer-probe sets are then loaded onto the microfluidic chip. Transcriptional analysis is performed for each cell across up to 96 genes in parallel, producing approximately 10,000 unique data points for each chip run. Figure reproduced with permission from (Glotzbach, Januszyk, 2011a).

Figure 2. Identification of distinct subgroups within a putatively homogeneous stem cell population

(A) Hierarchical clustering of 43 genes among 300 individual cells selected for low expression of CD34, a marker of hematopoietic stem cells. Gene expression is represented as fold change from the median on a color scale from yellow (high expression) to blue (low expression). Rows = gene targets, columns = individual cells. (B) Nine genes were identified that exhibit significantly different transcriptional patterns on single cell expression between populations of high expressing and low expressing CD34 hematopoietic stem cells. (C) Comparison of these subpopulations based on hierarchical clustering of the nine genes. Note the differential expression patterns of the two stem cell subpopulations based on CD34 expression. Figure reproduced with permission from (Glotzbach, Januszyk, 2011a).

Figure 3. Stem cell delivery methods

Commonly used methods to delivery stem cells include topical, injection, systemic, or scaffold-mediated approaches. Topical strategies are limited to sites that can be easily accessed, including cutaneous wounds or during surgical procedures. Injection therapies can access most anatomic sites, but stem cells are often delivered under hostile conditions (high pressures and cell densities). Systemic strategies require the ability of stem cells to properly home to damaged tissues and to effectively egress the circulation and enter the interstitial space. Scaffold-based techniques appear to be highly promising, offering the potential of a protective, three-dimensional vehicle with controlled spatial cues and precisely patterned matrix and cytokine gradients.

Figure 4. Biomaterial-enhanced expression of stemness genes in mesenchymal stem cells

(A) Reverse transcriptase polymerase chain reaction analysis of Oct4, Sox2, and Klf4 expression in mouse mesenchymal stem cells seeded into a carbohydrate-based dermal hydrogel compared to standard tissue culture plate. (B) Immunoblot protein validation and (C) quantification. (D) Immunofluorescence imaging demonstrates increased expression of stemness markers with seeding into a dermal hydrogel, suggesting enhancement of cell stemness via a biomaterial niche-driven approach. *p<0.05. Figure reproduced with permission from (Rustad, Wong, 2012).

Figure 5. Commercial development of a dermal hydrogel for stem cell-mediated wound healing

(A) Based on promising preclinical results, our laboratory is developing a dry hydrogel dressing that can be readily seeded with stem cells in solution. (B) Stem cells engrafted throughout the dermal-like matrix can be delivered directly into cutaneous wounds in a niche-like environment that has been demonstrated to promote wound vascularization and stem cell potency. (C) A topical adhesive covering is being developed to protect and secure the dermal hydrogel.