Microcarrier suspension cultures for high-density expansion and differentiation of human pluripotent stem cells to neural progenitor cells

Tissue Eng Part C Methods. 2013 Feb;19(2):166-80. doi: 10.1089/ten.TEC.2012.0146. Epub 2012 Sep 4.

Abstract

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×10⁶ cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×10⁶ cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes, showing that cells maintained their multilineage differentiation potential.

MeSH terms

  • Cell Differentiation*
  • Cell Division*
  • Cells, Cultured
  • Culture Media, Serum-Free
  • Flow Cytometry
  • Humans
  • Immunohistochemistry
  • Karyotyping
  • Neurons / cytology*
  • Pluripotent Stem Cells / cytology*
  • Real-Time Polymerase Chain Reaction

Substances

  • Culture Media, Serum-Free