Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling

Abstract

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.

Keywords: cGMP; cellular models of disease; cellular therapy; differentiation; drug discovery; glia; iPSCs; methods; neural stem cells; neurons; nomenclature.

Fig. 1

Phase-contrast comparison of classical PSC culture on MEFs with single-cell Accutase passaging and StemPro culture. A: PSCs (WA01.1-EI26-000.M8) mechanically passaged and transferred onto a feeder layer of irradiated MEFs showing classical PSC colony formation. ×4. B,C: Despite being seeded as single cells, Accutase-passaged cells quickly migrate to form small colonies with a great deal of obvious membranous material (Supp. Info. Video 1). B shows an early time (about 16 hr) after plating of PSCs (BG01.1-EI27-000.M6S6) continually passaged (six times) using Accutase and StemPro. ×40. C shows a later time (about 40 hr) after plating of PSCs (WA09.1-EI26-000.M8S16) continually passaged (16 times) using Accutase and StemPro. ×40. Scale bars = 500μm in A; 50 μm in B,C.

Fig. 2

Immunofluorescence staining of StemPro/Accutase/Matrigel-grown PSC cultures showing maintenance of pluripotency markers. BG01.1-EI27-000.M6S6 (A,B and WA09.1-EI26-000.M8S16 (C,D PSC cultures that were originally grown on MEFs and were transitioned to StemPro cultures with Accutase passaging. Cells were stained with a mouse monoclonal antibody to Oct-4 (A,C) or a goat polyclonal antibody to Sox-2 (B,D). ×40. Scale bars = 50μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig. 3

Chromosome spreads of Accutase-passaged StemPro PSC cultures. A: BG03.1-EI26-000.M2S20[46,XX], 20 StemPro passages without ROCK inhibitor. B: BG01.1-EI27-000.M6S20[46,XY], from S16 with ROCK inhibitor. C: WA09.1-EI26-000.M8S40[47,XX,+12], 40 StemPro passages without ROCK inhibitor.

Fig. 4

Staining of lentivirus- and Sendai virus-derived iPSCs from control and ASD cells. Clonal colonies with PSC morphology that stained strongly positive for Tra-1–60 (live staining) were picked for expansion, and colonies that showed the best morphology and homogeneity of staining with the PSC markers Nanog and Oct-4 were continuously expanded in culture. Phase-contrast images (A,C,E,I), DAPI staining (F,J), live Tra1–60 immunofluorescence (B,D), and immunocytochemical staining for Tra-1–60 (G), SSEA-4 (H), Nanog (K), and Sox-2 (L of SV-derived SC151.7-SF4-2I4.M5 iPSCs (A,B), LV-derived SC110.4-SF7-1I2.M8 iPSCs (C,D), LV-derived SC27.1-BN10-1I2.M4 iPSCs (E–H), and LV-derived SC30.1-BN10-1I3.M2 iPSCs (I–L) cultures. Scale bars = 50μm.

Fig. 5

Efficient, feeder-free, adherent-cell, defined differentiation of human ePSC and iPSC cultures to NSCs. NSC-differentiating ePSCs (BG03.1-EI26-000.M2S20-N1 [C–G,I,J]) and iPSCs (SC101.7-SF4-1I3.M10S5-N7 [A,B] and SC105.9-SF4-1I6.M24S5-N2 [H]) were characterized by immunofluorescence microscopy for the following markers: A, Oct-4; B, Pax-6; C, Forse-1; D, NCAM; E, Sox-1; F, N-CAD; G, nestin; H, CXCR-4; I, Olig-2; and J, β-tubulin. Scale bars = 50μm.

Fig. 6

Efficient, defined, chemical NSC induction from adherent, defined StemPro-adapted iPSC and ePSC cultures. Phase-contrast images showing the classical neural rosette morphology of NSCs derived from Accutase-passaged StemPro cultures of SC30.1-BN10-1I3.M2S4-N1 iPSCs (A following directed differentiation with Noggin and SB. ×10. WA09.1-EI26-000.M8S22-N2 ePSCs (B following directed differentiation with DMH1 and SB. ×10. BG03.1-EI26-000.M2S20-N2 ePSCs (C following directed differentiation with DMH1 and SB at higher magnification (×60) to show the resemblance to classical human brain-derived NSC cultures SC27.1-BN10 (Schwartz et al., 2003) as shown in D. ×40. Scale bars = 500μm in A,B; 50μm in C,D.

Fig. 7

Distribution of GFP- and/or mCherry-labeled cells after intracerebroventricular PSC injection [WA01.1-EI26-000.M32S21(mCh1), WA01.1-EI26-000.M32S21(GFP1); A,B] or NSC injection [WA01.1-EI26-000.M32S21(GFP1)-N7; C–H] in NSG mice. PSCs formed tumors in all cases examined. The tumors were almost always segregated by fluorophore (A,B; ×4), suggesting clonal expansion of only some of the injected cells. By contrast, PSC-derived NSCs never formed tumors or multicellular aggregates and appeared as single cells relatively evenly distributed in a variety of brain structures. C,D, E,F, and G,H show paired ×20 and ×60 images of frontal cortex, basal ganglia, and parietal cortex, respectively. Apical neurites can clearly be seen on some cells, suggesting neuronal differentiation. Scale bars = 500μm in A,B; 50μm in C–H.

Fig. 8

Process derived-NSCs can be terminally differentiated to neurons and glia. Immunofluorescence analysis of completely defined, small-molecule (DMH1 and SB)-directed NSC cultures that were either terminally differentiated to neurons (SC122.6-SF4-1I1M20S13-N2G8-3Nn80 [A], SC122.6-SF4-1I1M20S13-N2G12-4Nn60 [B]) in coculture with rat (A) or mouse (B) glia or terminally differentiated to astrocytes (SC171.7-SF5-1I1M6S12-N2G5-5A100; C). A is a composite image showing expression of the terminal neuronal markers synapsin (green) and MAP2ab (red) with nuclear counterstaining by DAPI (blue). B shows expression of the postsynaptic marker PSD-95 (red) on a biocytin (green)-injected neuron (this cell was subjected to electrophysiological recording as described in Fig. 9). C shows expression of the astrocyte-specific marker S100β (red) with nuclear counterstaining by DAPI (green). Scale bars = 50 μm.

Fig. 9

Process-derived NSCs yield functional neurons. A: Whole-cell voltage-clamp recording of a neuron derived from one line (SC122.6-SF4-1I1M20S13-N2G12-4Nn60) reveals a composite current with both inward sodium and outward potassium components in a neuron differentiated from human iPSC-derived NSCs. B: Current-clamp recording from the same neuron demonstrating the ability to fire multiple, overshooting action potentials in response to depolarizing current injection. C: Immunofluorescence analysis demonstrating that another recorded neuron filled with biocytin during whole-cell recording colabeled with the human-specific marker STEM121. C1: Phase-contrast image of recorded neuron. C2: Nuclear counterstaining with DAPI (blue). C3: Biocytin detection using a streptavidin-labeled fluorophore (red). C4: Colabeling of the biocytin-filled, electrically active neuron with the human-specific marker STEM121 (green).