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...

Tatiana, "The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels" (2011 AbstractSynthetic hydrogel scaffolds that can be used as culture systems that mimic the natural stem cell niche are of increased importance for stem cell biology and regenerative medicine. These artificial niches can be utilized to control the stem cell fate and will have potential applications for expanding/differentiating stem cells in vitro, delivering stem cells in vivo, as well as making tissue constructs. In this study, we synthesized hyaluronic acid (HA) hydrogels that could be degraded through a combination of cell-released enzymes and used them to culture mouse mesenchymal stem cells (mMSC). To form the hydrogels, HA was modified to contain acrylate groups and crosslinked through Michael addition chemistry using non-degradable, plasmin degradable or matrix metalloproteinase (MMP) degradable crosslinkers. Using this hydrogel we found that mMSC proliferation occurred in the absence of cell spreading, that mMSCs could only spread when both RGD and MMP degradation sites were present in the hydrogel and that mMSCs in hydrogels with high density of RGD (1000µM) spread and migrated faster and more extensively than in hydrogels with low density of RGD (100µM).