Human pluripotent stem cell (hPSC)-derived neural crest (NC) cells present a valuable tool for modeling aspects of human NC development, including cell fate specification, multipotency and cell migration. hPSC-derived NC cells are also suitable for modeling human disease and as a renewable cell source for applications in regenerative medicine. Here we provide protocols for the step-wise differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into neuroectodermal and NC cells using either the MS5 coculture system or a novel defined culture method based on pharmacological inhibition of bone morphogenetic protein and transforming growth factor-beta signaling pathways. Furthermore, we present protocols for the purification and propagation of hPSC-NC cells using flow cytometry and defined in vitro culture conditions. Our protocol has been validated in multiple independent hESC and hiPSC lines. The average time required for generating purified hPSC-NC precursors using this protocol is 2-5 weeks.
"Toxicol In Vitro 28(1):76–87 44. Lee JK, Jin HK, Bae JS (2010) Bone marrow-derived mesenchymal stem cells attenuate amyloid beta-induced memory impairment and apoptosis by inhibiting neuronal cell death. Curr Alzheimer Res 7(6):540–548 45. "
[Show abstract][Hide abstract] ABSTRACT: The developing brain is found highly vulnerable towards the exposure of different environmental chemicals/drugs, even at concentrations, those are generally considered safe in mature brain. The brain development is a very complex phenomenon which involves several processes running in parallel such as cell proliferation, migration, differentiation, maturation and synaptogenesis. If any step of these cellular processes hampered due to exposure of any xenobiotic/drug, there is almost no chance of recovery which could finally result in a life-long disability. Therefore, the developmental neurotoxicity (DNT) assessment of newly discovered drugs/molecules is a very serious concern among the neurologists. Animal-based DNT models have their own limitations such as ethical concerns and lower sensitivity with less predictive values in humans. Furthermore, non-availability of human foetal brain tissues/cells makes job more difficult to understand about mechanisms involve in DNT in human beings. Although, the use of cell culture have been proven as a powerful tool for DNT assessment, but many in vitro models are currently utilizing genetically unstable cell lines. The interpretation of data generated using such terminally differentiated cells is hard to extrapolate with in vivo situations. However, human umbilical cord blood stem cells (hUCBSCs) have been proposed as an excellent tool for alternative DNT testing because neuronal development from undifferentiated state could exactly mimic the original pattern of neuronal development in foetus when hUCBSCs differentiated into neuronal cells. Additionally, less ethical concern, easy availability and high plasticity make them an attractive source for establishing in vitro model of DNT assessment. In this review, we are focusing towards recent advancements on hUCBSCs-based in vitro model to understand DNTs.
"Lee et al. (2010) developed an hiPSC model of familial dysautonomia, a disorder that causes depletion of the autonomic and sensory neurons, induced by mutations in the IKBKAP gene. In the hiPSC system, they were able to correlate the loss of IKBKAP gene expression with defects in neurogenesis and diminished cell motility in hiPSC-derived neural crest cells. "
[Show abstract][Hide abstract] ABSTRACT: New advances in directing the neuronal differentiation of human embryonic and induced pluripotent stem cells (hPSCs, abbreviation intended to convey both categories of pluripotent stem cells) have promoted the development of culture systems capable of modeling early neurogenesis and neural specification at some of their critical milestones. The hPSC-derived neural rosette can be considered the in vitro counterpart of the developing neural tube, since both structures share a virtually equivalent architecture and related functional properties. Epigenetic stimulation methods can modulate the identity of the rosette neural progenitors in order to generate authentic neuronal subtypes, as well as a full spectrum of neural crest derivatives. The intrinsic capacity of induced pluripotent cell-derived neural tissue to self-organize has become fully apparent with the emergence of innovative in vitro systems that are able to shape the neuronal differentiation of hPSCs into organized tissues that develop in three dimensions. However, significant hurdles remain that must be completely solved in order to facilitate the use of hPSCs in modeling (e.g., late-onset disorders) or in building therapeutic strategies for cell replacement. In this direction, new procedures have been established to promote the maturation and functionality of hPSC-derived neurons. Meanwhile, new methods to accelerate the aging of in vitro differentiating cells are still in development. hPSC-based technology has matured enough to offer a significant and reliable model system for early and late neurogenesis that could be extremely informative for the study of the physiological and pathological events that occur during this process. Thus, full exploitation of this cellular system can provide a better understanding of the physiological events that shape human brain structures, as well as a solid platform to investigate the pathological mechanisms at the root of human diseases.
Frontiers in Neuroscience 07/2014; 8(8):183. DOI:10.3389/fnins.2014.00183 · 3.66 Impact Factor
"In the search of the ideal alternative to Schwann cells for peripheral nerve regeneration, many alternatives have been evaluated, especially in the field of stem cell research (Terenghi et al., 2009). Embryonic stem cells (ESC) (Cui et al., 2008; Ziegler et al., 2011), induced pluripotent stem cells (iPSC) (Lee et al., 2010; Kreitzer et al., 2013; Ikeda et al., 2014), and also mesenchymal adult stem cells (MSC) from various niches (that is bone marrow, fat, umbilical cord, dental pulp, skin) (McKenzie et al., 2006; Matsuse et al., 2010; Wakao et al., 2010; di Summa et al., 2011; Martens et al., 2014) have all been shown to be potential candidates as transplantable differentiated Schwann cell-like cells in nerve guidance tubes "
[Show abstract][Hide abstract] ABSTRACT: Injuries to peripheral nerves are common and cause life-changing problems for patients alongside high social and health care costs for society. Current clinical treatment of peripheral nerve injuries predominantly relies on sacrificing a section of nerve from elsewhere in the body to provide a graft at the injury site. Much work has been done to develop a bioengineered nerve graft, precluding sacrifice of a functional nerve. Stem cells are prime candidates as accelerators of regeneration in these nerve grafts. This review examines the potential of adipose-derived stem cells to improve nerve repair assisted by bioengineered nerve grafts.
Neural Regeneration Research 07/2014; 9(14):1341-6. DOI:10.4103/1673-5374.137585 · 0.22 Impact Factor
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