Advances in stem cell science have stimulated the prospect of stem cells as therapeutics. For the translation of stem cell research to technology, robust and efficient expansion and differentiation is essential. Rat multipotent adult progenitor cells (rMAPCs) are a type of adult stem cells isolated from the rodent bone marrow. MAPCs can be expanded in vitro without obvious senescence, and are capable of differentiating into cell types of mesoderm, endoderm, and ectoderm in vitro. They are typically maintained surface adherent at low cell densities of 100-300 cells/cm2 in order to maintain their broad potency, which would make scale-up for further clinical applications cumbersome. In this study, we explored different cultivation methods to investigate the feasibility of scalable culture systems for rMAPCs: (1) high density 2D culture, stirred bioreactor culture as (2) 3D aggregates and (3) on microcarriers.
Culturing cells under hypoxic condition (5% O2) during the isolation, has yielded rMAPC expressing high levels of the embryonic stem cell specific transcription factor Oct4, which is associated with their greater potency. First, the effect of oxygen tension and cell density on the growth rate and potency of MAPC were examined. MAPC exhibited an increased growth rate at hypoxic conditions (5%) than at normoxic conditions (21%). Furthermore, when inoculated at a cell density of 1000 cells/cm2, MAPC exhibited a small but significant increase in growth rate compared to cells seeded at 300 cells/cm2 at both oxygen levels, though the difference was more pronounced under hypoxic conditions. The Oct4 mRNA or protein expression level and the ability of MAPC to differentiate towards endothelium- and hepatocyte-, and neuroectoderm-like cells were shown to be unaffected by cultivation at a higher cell density and/or oxygen tension for 48 days (1000 cells/cm2; 21% O2; with subculturing every 48 hr). The results provide evidence that MAPC isolated under hypoxic conditions and expressing high levels of Oct4 can be readily cultured at a higher cell density without any apparent loss of potency.
Encouraged by MAPCs ability to grow at high density, we explored aggregate formation of MAPC for cell expansion as well as differentiation. Culture in aggregates may be an ideal method to allow large scale expansion, if combined with bioreactor cultures. Time lapse microscopy revealed three stages during the initial period of aggregate formation: agglomeration, compaction, and expansion. Compared to cells from adherent culture, significantly more cells from 3D culture are in G0/G1 phase and fewer in S phase suggesting a partial restriction in cell proliferation possibly due to spatial restriction in aggregates. There was no significant difference in Oct4 level and aggregate size when aggregation was at 5% or 21% O2 after 4 day culture. However, aggregation at 21% O2 increased the percent of cells in G0/G1 and increased expression of early differentiation markers such as Flk1 and Afp. Cultivation of MAPC aggregates in stirred bioreactor lead to a 70-fold expansion in six days with final cell densities of about 106 cells/ml. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed Albumin, Aat, Tat transcripts and also secreted albumin and urea.The cells expressed several mature hepatocyte-lineage genes and asialoglycoprotein receptor-1 (ASGPR-1) surface protein, and secreted albumin and urea. Lastly, the experience with MAPC microcarrier culture was extended to human embryonic stem cell (hESC) microcarrier culture. hESCs as small clumps were attached to Matrigel-coated microcarriers and expanded 10-fold during 4 day static culture. The level of pluripotency-related genes, OCT4 and SOX2, were maintained compared to day 0 cells. The cells expanded on microcarriers underwent hepatic differentiation to increase hepatic genes such as AFP and ALBUMIN.
Both aggregate and microcarrier cultivation methods for scalable expansion combined with differentiation can potentially be used to generate large numbers of MAPC and MAPC-derived differentiated cells. These culture systems thus offer the potential of large-scale expansion and differentiation of stem cells in a more controlled bioreactor environment.
University of Minnesota Ph.D. dissertation. May 2010. Major: Biomedical Engineering. Advisors: Wei-Shou Hu and Catherine Verfaillie. 1 computer file (PDF); xiv, 184 pages, appendix p. 182-184. + 1 computer file (WMV); stop motion video clip titled MAPCaggregation.
Scalable culture systems for expansion and directed differentiation of rat multipotent adult progenitor cells..
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