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.

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

“…2. Coat fresh plates or flasks with ECM, as previously described 9,10 . Some ECMs -such as those derived from mouse sarcoma cell lines -are only liquid between 4 and 15 °C, so use a sterilized cold block or gel ice pack to keep the ECM cold while working in the heated process chamber.…”

“…14. If cryopreservation of the cells is required, follow the appropriate protocol 9,10 , but ensure that all supplies and freezing media are prepared in advance and placed inside the process chamber. DMSO is very toxic to cells at 37 ºC, so work very quickly.…”

“…A cell production facility that can address both contamination risks and gas concentrations and that can be qualified to meet cGMP criteria 9 provides high quality cells for basic science research as well as clinical applications 1,6,7 . Such a cell production facility consists, at a minimum, of the following components: a process chamber, which acts as a heated workspace for the feeding and manipulation of cell cultures; a laminar flow hood, for the initial sterilization of reagents, tubes, and tools; two buffering airlock chambers in between the hood and the process chamber; two cell culture incubators accessible from the process chamber; a microscope chamber adjacent to the process chamber; and finally, computer software to set and monitor the conditions within these modules.…”

The potential of induced pluripotent stem cells in models of neurological disorders: implications on future therapy

“…In spite of the above mentioned challenges, improvements for iPSC differentiation to neural cells and tissues are being made through the development of better defined, optimised and efficient protocols[30, [99][100][101][102][103], bolstered by increased availability of superior stem cells attributable to improved somatic cell reprogramming, stem cell culture, banking and distribution [104][105][106][107]. A major advance from traditional differentiation methods is the circumvention of embryoid body (EB) formation for more efficient and direct induction of neural progenitor cells (NPCs) and expansion of neurospheres [99][100][101][102].…”

“…A major advance from traditional differentiation methods is the circumvention of embryoid body (EB) formation for more efficient and direct induction of neural progenitor cells (NPCs) and expansion of neurospheres [99][100][101][102]. For example, Lie et al proffers high yield production of NPCs from feeder-free iPSC aggregates cultured in mTeSR™1(Stem Cell Technologies) [99].…”