Defining cell-matrix combination products in the era of pluripotency

Abstract

Human pluripotent stem (hPS) cells provide an attractive opportunity for the manufacture of a wide array of therapeutic cell types. The challenges to commercialization include the thousand-fold diversity of cell types emerging from hPS cells and the associated difficulties in validating processes to reliably generate cells with precise identity and purity. Improved methods of controlling the dosage and migration of hPS-derived cells in solid tissues are also needed. To directly address these issues, we clonally expanded proliferating lineages of cells that were intermediate in regard to their state of differentiation between hPS and terminally differentiated cells. These cells called monoclonal embryonic progenitors (hEP), are expandable mortal lineages with diverse site-specific homeobox gene expression and multipotentiality. In this review, we discuss methods of generating combination products wherein the fate space of precisely identified monoclonal hEP cells is mapped by differentiating the cells in vitro in HyStem-3D bead arrays in the presence of diverse growth factors. This combination of discovery processes has the potential to translate directly into cell-matrix formulations that can be used to generate pre-clinical data leading to human clinical trials and potentially new medical therapies.

Keywords: cartilage; differentiation; embryonic progenitor cells; embryonic stem cells; hyaluronic acid; matrices; tissue engineering.

Figure 1. Heterogeneous differentiation of hPS cells vs. the clonal expansion of hEPs as a means of manufacturing therapeutic products. (A) With heterogeneous differentiation, hPS cells such as hES cells are scaled in the undifferentiated immortal state, exposed to a differentiation protocol, and the desired cell type is purified from the heterogeneous mixture. (B) Clonal lines are generated from partially differentiated hPS cells, the clonal lines are expanded in a mortal state, then terminally differentiated for research or therapeutic use.

Figure 2. Phase-contrast photographs of a representativ hEP cell line micromass and HyStem-4D bead used in fate-space screening A. hEP cell micromass from the cell line T42 cultured in the presence of BMP4. B. hEP cell HyStem-4D bead constructs from the cell line T42 cultured in the presence of BMP4. (Scale bar, 100 microns).

Figure 3. Strategy of fate-space screening. Diverse clonal hEP cell lines are cultured in high density conditions such as micromasses or HyStem-4D bead arrays in the presence of diverse differentiation conditions such as physiological concentrations of TGFβ family members, FGFs, retinoic acid, and modulators of Wnt signaling. After 14–21 d, RNA is analyzed by gene expression microarrays.

Figure 4. Representative histology of differentiated HyStem-C constructs in vitro and tracking of human hEP-derived differentiated cells in HyStem-C in tissue sections. The hEP cell line E15 (P19) was differentiated for 14 d in HyStem-C in vitro supplemented with TGFβ3 and BMP7. (A) H&E staining of differentiated E15/HyStem-C construct, (B) Safranin-O staining of differentiated E15/HyStem-C construct, (C) The cell line 4D20.8 was formulated in HyStem-C and implanted in vivo into a rat femoral defect for four weeks and transplanted cells were localized with anti-human mitochondrial antibody and H&E staining (red arrows).

Figure 5. Relative expression levels of MYH11, FABP4, DCN, and TIMP4 in hEP cell lines differentiated in BMP4 in both micromass and HyStem-4D bead arrays. Expression levels of MYH11 (solid black), FABP4 (stippled), DCN (gray) and TIMP4 (cross-hatched) are shown for the hEP cell lines E15, E69, T42, and W10 in both micromass and HyStem-4D bead arrays both being supplemented with BMP4. (RFUs, relative fluorescence units; RFU values of < 100 considered as background signal). Cntl, Control; MM, micromass; HS, HyStem-C constructs.