Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions

Kirkeby, Agnete; Grealish, Shane; Wolf, Daniel; Nelander, Jenny; Wood, James; Lundblad, Martin; Lindvall, Olle; Parmar, Malin

doi:10.1016/j.celrep.2012.04.009

Cited by 605 publications

(703 citation statements)

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“…3 D-F). Despite a similar degree of morphological maturation at a time in which a substantial fraction of both grafted fetal cells and human embryonic stem cells express TH (21), few of the hiN cells obtained via conversion in vivo expressed TH at this stage (Fig. 3G).…”

Section: Resultsmentioning

confidence: 98%

Generation of induced neurons via direct conversion in vivo

Torper

Pfisterer

Wolf

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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376

319

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Cellular reprogramming is a new and rapidly emerging field in which somatic cells can be turned into pluripotent stem cells or other somatic cell types simply by the expression of specific combinations of genes. By viral expression of neural fate determinants, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons. The resulting cells are nonproliferating and present an alternative to induced pluripotent stem cells for obtaining patient-and disease-specific neurons to be used for disease modeling and for development of cell therapy. In addition, because the cells do not pass a stem cell intermediate, direct neural conversion has the potential to be performed in vivo. In this study, we show that transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, convert into neurons when reprogramming genes are activated after transplantation. Using a transgenic mouse model to specifically direct expression of reprogramming genes to parenchymal astrocytes residing in the striatum, we also show that endogenous mouse astrocytes can be directly converted into neural nuclei (NeuN)-expressing neurons in situ. Taken together, our data provide proof of principle that direct neural conversion can take place in the adult rodent brain when using transplanted human cells or endogenous mouse cells as a starting cell for neural conversion.T he ability to reprogram somatic cells to pluripotent stem cells or other somatic cell types by expressing key combinations of genes has opened up new possibilities for disease modeling and cell therapy (1, 2). Using this technique, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons (iNs), using viral delivery of the three neural conversion factors achaete-scute complex-like 1 (Ascl1), brain-2 (Brn2a), and myelin transcription factor-like 1 (Myt1l) (ABM) (3, 4). A growing number of studies now show that by altering the combination of genes used for reprogramming, different subtypes of neurons are obtained (3,5,6). Importantly, the resulting cells are nonproliferating, which makes them an interesting alternative to induced pluripotent stem cells as a source of patient-specific neurons for cell replacement therapy, once efficient grafting strategies for these cells are developed.The adult brain has a very limited inherent capacity for repair, and new neurons are only formed in two discrete regions: the subventricular zone of the lateral ventricles, which generates neurons migrating to the olfactory bulb, and the hippocampus (7,8). Experimental studies have shown that these endogenous progenitors can also be recruited to generate new neurons in other regions as well in response to injury (9-11). However, the number of new neurons is very low, their migration is hard to control, and the therapeutic implications are unclear. Several cell types residing outside the neurogenic niche, such as parenchymal...

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Section: Resultsmentioning

confidence: 98%

Generation of induced neurons via direct conversion in vivo

Torper

Pfisterer

Wolf

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

376

319

View full text Add to dashboard Cite

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“…7D). Finally, these DA progenitors could subsequently be cultured on 2D surfaces as described (37) for differentiation into mature TH+ DA neurons (Fig. 7E).…”

Section: Resultsmentioning

confidence: 99%

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

Lei

Schaffer

2013

Proc. Natl. Acad. Sci. U.S.A.

294

312

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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...

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“…Human embryonic stem cells provide new possibilities for the clinical treatment of a number of diseases such as diabetes mellitus type 1 [1], Parkinson's disease [2], Huntington's disease [3], cardiac failure [4], etc. In addition, the ability of pluripotent human embryonic stem cells to differentiate into various cell types enables us to utilize them for studying early human development and provides a cell source for cellular model systems.…”

Section: Introductionmentioning

confidence: 99%