Biomimetic Three-Dimensional Cultures Significantly Increase Hematopoietic Differentiation Efficacy of Embryonic Stem Cells

Abstract

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation, we cultured mouse ES cells on 3D, highly porous, biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however, unlike 2D differentiation, these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly, the efficiency of hematopoietic precursor cell (HPC) generation on 3D, as indicated by the expression of various HPC-specific surface markers (CD34, Sca-1, Flk-1, and c-Kit) and colony-forming cell (CFC) assays, was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs.

Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

“…Efficacy of HSPC generation was analyzed as reported before. 25 15,16 In addition, cells that express only c-Kit or only sca-1 were also analyzed to determine the unique profile of differentiated cells in each bioreactor culture.…”

“…[22][23][24] Our laboratory has previously demonstrated that this type of bioreactor could significantly increase hematopoietic differentiation of cells cultured in scaffolds. 25 Similarly, the SyntheconÔ rotary cell culture system, a microgravity type bioreactor with a slowturning lateral vessel, originally developed by NASA, has also been reported to increase the efficiency of EB formation and differentiation of stem cells into the three germ layers of embryonic development. 26 Comparison of spinner flask and Synthecon bioreactors for human ES cell differentiation, specifically focusing on cardiac and endothelial lineages, has also been recently reported.…”

Bioreactor development for stem cell expansion and controlled differentiation

“…However, several groups have recently shown that mouse EBs can be formed directly from enzymatically dissociated mESCs in stirred reactors [19••] or a rotary cell culturing system [20]. Mouse EBs can also be formed in stirred vessels by removing LIF from mESC aggregates produced in the same vessels [16••] [22].…”

Three‐ and four‐dimensional visualization of cell migration using optical coherence tomography

“…Cell behavior and phenotype have been shown to differ in 2D and 3D environments [22]. Although valuable insights into cell migration have been gained from 2D studies into cell movement, cells in vivo undergo morphogenesis and migrate in 3D.…”