A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

Abstract

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human-or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 10 72 -fold expansion over 280 d), yield (∼2.0 × 10 7 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 10 7 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.H uman pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) (1) and induced pluripotent stem cells (iPSCs) (2), have the capacities for indefinite in vitro expansion and differentiation into all cell types within adults (3). They therefore represent highly promising cell sources for numerous biomedical applications, such as cell replacement therapies (4, 5), tissue and organ engineering (6), and pharmacology and toxicology screens (7,8). However, these applications require large numbers of cells of high quality (4, 6-8). For instance, ∼10 5 surviving dopaminergic (DA) neurons, ∼10 9 cardiomyocytes, or ∼10 9 beta cells are likely required to treat a patient with Parkinson disease (PD), myocardial infarction (MI), or type I diabetes, respectively (9). Additionally, far more cells are needed initially because both in vitro cell culture yields and subsequent in vivo survival of transplanted cells are typically very low. As examples of the latter, only ∼6% of transplanted dopaminergic neurons or ∼1% of injected cardiomyocytes reportedly survive in rodent models several months after transplantation (10, 11). Furthermore, there are large patient populations with degenerative diseases or organ failure (9), including over 1 million people with PD, 1-2.5 million with type I diabetes, and ∼8 million with MI in the United States alone (12). Large numbers of cells are also necessary for applications such as tissue engineering, where for ex...

Scalable culture of human pluripotent stem cells in 3D

“…Moreover, the 3D hydrogel environments might also be conducive to enhance strategies for direct reprogramming of somatic cells to neural lineages and functional cell types (18)(19)(20). Only time will tell how much 3D scalable suspension culture techniques will ultimately impact hPSC growth and differentiation protocols, but it appears likely that their use for such purposes will "expand" in the near future based on the encouraging results of this study reported in PNAS (7).…”

“…Small molecules, such as Rho kinase inhibitor, are commonly added during hPSC passaging in an attempt to inhibit cell death, and were used by the authors in their final optimized protocol because it similarly improved single-cell survival in the 3D hydrogels. Seeding of the single cells dispersed evenly throughout the hydrogel matrix enabled growth of cells under nearly identical biochemical and biophysical conditions, and different cell lines behaved similarly in the 3D environments (7). The similarities in the size and appearance of the hPSC clusters in the hydrogels suggested better synchronized growth of the overall culture.…”

“…In PNAS, Lei and Schaffer address many of these technical challenges with their report of a unique 3D culture system for scalable and efficient hPSC expansion and differentiation that is completely defined and capable of being compatible with good manufacturing practices (7). An important trait of the synthetic polymer hydrogel system chosen by Lei and Schaffer is that it is thermoresponsive, thereby enabling simple encapsulation and rapid retrieval of hPSCs at any time by switching the temperature between 4°C and 37°C, based on the phase transition behavior of the poly(N-isopropylacrylamide)-co-poly(ethylene glycol) (PNIPAAm-PEG) hydrogel going from a liquid to a solid gel as the temperature is increased.…”

Usefulness of the Behavior of Fibroblast Attachment to Coils in Thermoreversible Gelation Polymer for Aneurysmal Coil Treatment

“…The thermoreversible gelation polymer (TGP) is an aqueous solution that remains liquid at temperatures less than the sol-gel transition temperature (TT) and becomes a gel at temperatures higher than the TT. TGP was originally developed as a cell culture medium [14,15] and can be used as a drug delivery vehicle for chemotherapy [16,17] or active substance carrier for basic fibroblast growth factor together with live fibroblasts [18]. Our group has already reported TGP as a new thrmoreversible liquid embolic agent for treatment of experimental aneurysms [18][19][20][21].…”

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

“…to simplify process and facilitate scale-up from laboratory-based research to industrial-scale biomedicine) and incorporate specialist media, for example mTeSR™ [110,111] Although there have been few reports to date of biomaterial based iPSC culture for neural induction, the ability for materials to interface with hESCs for neural differentiation is indubitably applicable. Nevertheless, a recent landmark report involving both hESCs and iPSCs describes 3-D poly(N-isopropylacrylamide)-copoly(ethylene glycol) (PNIPAAm-PEG) hydrogel support of stem cell expansion and differentiation [125] (Table 1). PNIPAAm-PEG is a synthetic thermoresponsive hydrogel that is liquid at low temperature for cell loading, which solidifies at 37 o C for subsequent for in situ differentiation, we will progress to more complex multicellular printing and placement for more efficient and germane construct design, with the holy-grail for developing multidimensional "live" constructs being able to support vascularisation towards formation of clinical-scale tissues and whole-organ substitutes [130].…”