Derivation of mesenchymal stromal cells from pluripotent stem cells through a neural crest lineage using small molecule compounds with defined media

Abstract

Neural crest cells (NCCs) are an embryonic migratory cell population with the ability to differentiate into a wide variety of cell types that contribute to the craniofacial skeleton, cornea, peripheral nervous system, and skin pigmentation. This ability suggests the promising role of NCCs as a source for cell-based therapy. Although several methods have been used to induce human NCCs (hNCCs) from human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), further modifications are required to improve the robustness, efficacy, and simplicity of these methods. Chemically defined medium (CDM) was used as the basal medium in the induction and maintenance steps. By optimizing the culture conditions, the combination of the GSK3β inhibitor and TGFβ inhibitor with a minimum growth factor (insulin) very efficiently induced hNCCs (70-80%) from hPSCs. The induced hNCCs expressed cranial NCC-related genes and stably proliferated in CDM supplemented with EGF and FGF2 up to at least 10 passages without changes being observed in the major gene expression profiles. Differentiation properties were confirmed for peripheral neurons, glia, melanocytes, and corneal endothelial cells. In addition, cells with differentiation characteristics similar to multipotent mesenchymal stromal cells (MSCs) were induced from hNCCs using CDM specific for human MSCs. Our simple and robust induction protocol using small molecule compounds with defined media enabled the generation of hNCCs as an intermediate material producing terminally differentiated cells for cell-based innovative medicine.

Figure 1. Induction of p75^high cells from hPSCs.

A) Schematic representation of the protocol. B) Morphology of colonies during the induction. Phase contrast images were taken on days 0, 3, and 7. Scale bar, 200 µm. C) The fraction of p75-positive cells in 201B7 cells was treated with SB431542 (SB) (10 µM) and CHIR99021 (CHIR) (indicated concentration) for seven days, stained with an anti-p75 antibody, and analyzed by FACS. D) Fraction of the p75^high population induced by SB (10 µM) and CHIR (1 µM) from hESCs (KhES1, KhES3, H9) and hiPSCs (414C2, 201B7). Average ± SD. N = 3, biological triplicate. E) Immunocytochemical analyses of colonies on day 7 (201B7). Cells were stained with antibodies against PAX6, TFAP2A, and p75. Scale bar, 100 µm.

Figure 2. Expression profiles of sorted p75^high cells.

A) The expression of marker genes in sorted p75^high and p75^low cells. The mRNA expression of each gene was analyzed by RT-qPCR in undifferentiated 201B7 (hiPSCs) and sorted p75^low and p75^high cells, and was shown as a relative value using the level in sorted p75^high cells as 1.0. Average ± SD. N = 3, biological triplicates. B) Clustering analyses of NCC markers in p75^high populations from several hESC and hiPSC lines. Marker genes for each sub-population of NCCs were labeled using the indicated colors.

Figure 3. Sustained expansion of hNCCs with original characteristics.

A) Schematic representation of the culture conditions. B) Growth profile of 201B7-derived hNCCs. Average ± SD. N = 3, biological triplicate. C) Phase contrast images and immunostaining of TFAP2A in 201B7-derived hNCCs at PN0 and PN4, Scale bar, 200 µm. D) Hierarchical clustering analyses of hPSCs and hPSC-derived hNCCs at PN0 and PN10.

Figure 4. Modulation of regional characteristics of hNCCs.

A) Schematic representation of culture conditions for the induction and maintenance of hNCCs. RA, retinoic acid (100 nM). B) Schematic distribution of marker-positive cells in the murine embryo. DI, diencephalon; MB, midbrain; BA1 to BA4, branchial arch 1 to branchial arch 4; r1 to r6; rhombomere 1 to rhombomere 6. C) The mRNA expression of regional specifier genes in hNCCs. p75^high cells were collected at the end of the hNCC induction by FACS and seeded onto fibronectin-coated dishes. RNAs were extracted when cells reached a semi-confluent state and used for RT-qPCR. The relative expression level of each gene was demonstrated using the value of cells cultured in CDM (OTX2 and DLX1) or CDM + RA (HOXA2 and HOXA3) as 1.0. Average ± SD. N = 3, biological triplicate.

Figure 5. Derivation of peripheral neural cells, glia, and melanocytes from hNCCs.

A) Neuronal differentiation of 201B7-derived hNCCs. Cells were stained with an antibody against peripherin (red) and Tuj-1 (green). B) The glial differentiation of 201B7-derived hNCCs. Cells were stained with an antibody against GFAP. Scale bar, 50 µm. C) Melanocyte differentiation of 201B7-derived hNCCs. The mRNA expression levels of MITF and c-KIT genes were shown as a relative value using the value in 201B7-derived hNCCs and 201B7 as 1.0, respectively. Average ± SD. N = 3, biological triplicates.

Figure 6. Derivation of corneal endothelial cells from hNCCs.

A) Schematic protocol for the induction of corneal endothelial cells. B) Phase contrast images of cells before (D8) and after (D19) the induction. Scale bar, 200 µm. C) The expression of ZO-1 in cells at D12. Cells were stained with an antibody against ZO-1. D) The mRNA expression of corneal endothelial cell marker genes. RNAs were extracted from cells at D10, D12, and D15. The expression level of each gene was demonstrated as a relative value using the value in human primary corneal endothelial cells as 1.0. Average ± SD. N = 3, technical triplicate. We performed this CEC induction twice and confirmed its reproducibility.

Figure 7. Derivation of hMSCs from hNCCs.

A) Schematic protocol for the induction of hMSCs. B) Phase contrast images of cells before (D8) and after (D21) the induction. Scale bar, 200 µm. C) Expression of surface markers in hBM-MSCs (hBM90) and 201B7-derived MSCs (201B7-MSC). D) Hierarchical clustering analyses by genome-wide gene expression profiles. RNAs were extracted from hBM-MSCs (BM90, BM91 and BM94), induced-MSCs, and the corresponding hNCCs and hiPSCs. E) Differentiation properties of induced-MSCs. The induction for osteogenic (OI), chondrogenic (CI), and adipogenic (AI) lineages was performed as described in the Materials and Methods section and evaluated by Alizarin Red staining (OI), Alcian Blue staining (CI), and Oil Red O staining (AI), respectively. Scale bar, 100 µm. F) Population of SSEA4-positive cells. G) The expression levels of pluripotent markers (OCT3/4, NANOG and SOX2) in hPSCs, hNCCs, and hMSCs. Average ± SD. N = 3, biological triplicates.

Figure 8. Derivation of hMSCs from hNCCs under defined culture conditions.

A) Schematic protocol for the induction of hMSCs from hNCCs under defined culture conditions. B) Phase contrast images of cells 0, 7, and 21 days after the hNCC and hMSC induction, respectively. Scale bar, 200 µm. C) Expression of hMSC-related surface markers in hMSCs induced under defined culture conditions. D) Osteogenic differentiation (OI) properties of hMSCs induced under defined culture conditions. hMSCs were cultured during the induction period in STK2 as a control.