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.

Human embryonic stem cells (hESC) are characterized by their ability to self-renew and differentiate into all cell types of the body, making them a valuable resource for regenerative medicine. Yet, the molecular mechanisms by which hESC retain their capacity for self-renewal and differentiation remain unclear. The Hedgehog signaling pathway plays a pivotal role in organogenesis and differentiation during development, and is also involved in the proliferation and cell-fate specification of neural stem cells and neural crest stem cells. As there has been no detailed study of the Sonic hedgehog (SHH) signaling pathway in hESC, this study examines the expression and functional role of SHH during hESC self-renewal and differentiation. Here, we show the gene and protein expression of key components of the SHH signaling pathway in hESC and differentiated embryoid bodies. Despite the presence of functioning pathway components, SHH plays a minimal role in maintaining pluripotency and regulating proliferation of undifferentiated hESC. However, during differentiation with retinoic acid, a GLI-responsive luciferase assay and target genes PTCH1 and GLI1 expression reveal that the SHH signaling pathway is highly activated. Besides, addition of exogenous SHH to hESC differentiated as embryoid bodies increases the expression of neuroectodermal markers Nestin, SOX1, MAP2, MSI1, and MSX1, suggesting that SHH signaling is important during hESC differentiation toward the neuroectodermal lineage. Our findings provide a new insight in understanding the SHH signaling in hESC and the further development of hESC differentiation for regenerative medicine.

The utilization of human embryonic stem cells (hESC) in regenerative medicine largely depends on the development of technologies that will allow efficient genetic manipulation of the cells in vitro. Although a few studies have described the transfection of hESC for generation of reporter lines stably expressing specific transgenes driven by different promoters, the optimal choice of promoter system for driving transgene in hESC has yet to be elucidated. We show for the first time that Chinese hamster elongation factor-1 alpha (CHEF1) promoter robustly drove reporter gene expression higher than the human elongation factor 1 alpha (hEF1 alpha), other constitutive Chinese hamster promoters, human cytomegalovirus (CMV) immediate early enhancer/promoter and SV40 promoters in hESC by quantitative analysis. We also successfully generated stably transfected hESC lines using this CHEF1 promoter system and demonstrated that they continued to express enhanced green fluorescent protein (EGFP) during prolonged undifferentiated proliferation, in differentiated embryoid bodies (EBs), and in teratomas without transgene silencing. By immunofluorescence staining and D ow cytometry analysis, the pluripotent markers, OCT-4, SSEA-4, and TRA-1-60, continued to be expressed in undifferentiated CHEF1-EGFP expressing hESC lines. When the stably transfected hESC were directed to differentiate into neural precursors in vitro, high-level EGFP expression was maintained and co-expression of neural markers, Nestin, and beta-tubulin III was observed. The morphology, karyotype, and telomerase activity of CHEF1-EGFP expressing hESC were normal after >50 continuous passages, and the cells retained the ability to differentiate into derivatives of the three germ layers in vitro as confirmed by RT-PCR analysis and immunocytochemical staining and in vivo teratoma formation. Therefore, stable CHEF1-EGFP hESC lines retained the capability for self-renewal and pluripotency. The novel CHEF1 promoter system described here enables high-level transgene expression in the stably transfected hESC. It may have signi, cant implication for uses in bioprocess development and future development of gene-modified hESC in tissue regeneration and transplantation applications.