Rapid and Efficient Generation of Functional Motor Neurons From Human Pluripotent Stem Cells Using Gene Delivered Transcription Factor Codes

Hester, Mark E.; Murtha, Matthew; Song, SungWon; Rao, Meghan; Miranda, Carlos J.; Meyer, Kathrin; Tian, Jin Bin; Boulting, Gabriella L.; Schaffer, David V.; Zhu, Michael X.; Pfaff, Samuel L.; Gage, Fred H.; Kaspar, Brian K.

doi:10.1038/mt.2011.135

Cited by 167 publications

(141 citation statements)

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“…Importantly, initiation of RA treatment at day 3 followed by subsequent neuronal maturation also produced a high percentage of HB9 þ cells (60.1 ± 9.5%; n ¼ 8) from H9 hESCs, suggesting conserved responses in hESCs. Next, we determined whether the hESC-derived MNs were functional using established assays 8,[26][27][28] . First, the cells were capable of extending long projections, suggesting neuronal maturation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Despite these encouraging advances, limitations remain in the existing MN protocols that hamper the use of human pluripotent stem cells. For example, the differentiation procedures as they stand are tedious, time consuming (up to 2 months), requires genetic manipulations and repeated viral infections, and are of low efficiencies (10-40%) and yields 7,8 . Furthermore, the differentiation media and substrates contain undefined factors, animal feeder cells and/or animal products and are therefore not clinically compatible.…”

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confidence: 99%

“…Considerable progress has been made in the past decade in differentiating human pluripotent stem cells into MNs. A number of protocols have been developed, which utilize various themes including co-culture with stromal feeders (PA6 or MS5) 6 , embryoid body (EB) induction followed by neural rosette formation, neural patterning and neuronal maturation 7 , and neural progenitor induction from EBs followed by genetic programming using adenovirus-mediated gene delivery 8 . Despite these encouraging advances, limitations remain in the existing MN protocols that hamper the use of human pluripotent stem cells.…”

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confidence: 99%

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High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1

et al. 2014

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Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development, modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (B70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (4250%) MN production in chemically defined adherent cultures. Furthermore, we show that Islet-1 is essential for formation of mature and functional human MNs, but, unlike its mouse counterpart, does not regulate cell survival or suppress the V2a interneuron fate. Together, our discoveries improve the strategy for MN derivation, advance our understanding of human neural specification and MN development, and provide invaluable tools for human developmental studies, drug discovery and regenerative medicine.

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Section: Resultsmentioning

confidence: 99%

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High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1

et al. 2014

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“…This combinatorial action of Lhx3 and Isl1 in MN generation provides a useful model to test the strategy to differentiate stem cells to a specific neuronal type by expressing a defined set of transcription factors. Indeed, the coexpression of Isl1 and Lhx3, along with other transcription factors that induce neurogenesis, is capable of directing differentiation of ESCs, iPSCs, and fibroblasts to MNs (27,28). However, a major challenge remains that each individual transcription factor has its own activity in cell lineage determination, distinct from a combinatorial function, and thus the expression ratio of the transcription factors should be optimized.…”

Section: Discussionmentioning

confidence: 99%

Fusion protein Isl1–Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs

Lee

Cuvillier

Lee

et al. 2012

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

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Combinatorial transcription codes generate the myriad of cell types during development and thus likely provide crucial insights into directed differentiation of stem cells to a specific cell type. The LIM complex composed of Isl1 and Lhx3 directs the specification of spinal motor neurons (MNs) in embryos. Here, we report that Isl1-Lhx3, a LIM-complex mimicking fusion, induces a signature of MN transcriptome and concomitantly suppresses interneuron differentiation programs, thereby serving as a potent and specific inducer of MNs in stem cells. We show that an equimolar ratio of Isl1 and Lhx3 and the LIM domain of Lhx3 are crucial for generating MNs without up-regulating interneuron genes. These led us to design Isl1-Lhx3, which maintains the desirable 1:1 ratio of Isl1 and Lhx3 and the LIM domain of Lhx3. Isl1-Lhx3 drives MN differentiation with high specificity and efficiency in the spinal cord and embryonic stem cells, bypassing the need for sonic hedgehog (Shh). RNA-seq analysis revealed that Isl1-Lhx3 induces the expression of a battery of MN genes that control various functional aspects of MNs, while suppressing key interneuron genes. Our studies uncover a highly efficient method for directed MN generation and MN gene networks. Our results also demonstrate a general strategy of using embryonic transcription complexes for producing specific cell types from stem cells. D eveloping central nervous system (CNS) produces a vast number of neuronal types, but adult CNS has only limited capacity to regenerate neurons. This has prompted great interest in identifying methods to produce specific neuronal types from stem cells. Production of differentiated cell types from pluripotent stem cells, such as embryonic stem cells (ESCs), should enable a continuous supply of diseased cell types for drug screening and cell replacement therapy and provide valuable insights into the pathophysiology of human diseases. One important challenge in this effort is to steer stem cells into specific cell types. Recapitulation of normal developmental processes using embryonic inductive signals has been used to drive differentiation of pluripotent stem cells into specific cell types (1). However, this strategy tends to trigger formation of mixed cell types rather than a targeted cell type, because each inductive signal is used in multiple developmental pathways. This shortcoming might be circumvented by using more specific, downstream transcription factors of inductive signals. In this regard, it should be noted that many transcription factors function in combination to determine cell fates during development, suggesting that coexpression of multiple transcription factors could be a more effective method to generate a particular cell type from pluripotent stem cells.Motor neurons (MNs) in the spinal cord project axons to muscles and control their contraction. The developmental pathways to generate MNs have been relatively well studied. In the developing spinal cord, sonic hedgehog (Shh) signal triggers the expression of two LIM homeodomain...

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“…Table 2 illustrates several types of iPSC-based physiological models, in which iPSCs may be used to recapitulate neural cell interactions with other cells and their environment. Several functional interactions can be assessed [48][49][50][51][52][53][54]; these include modeling (1) blood-brain barrier, (2) synaptic networks, (3) neuron-astrocyte interactions, (4) oligodendrocyte myelination, (5) peripheral myelination, (6) retinal processes, (7) developmental processes, and (8) immunological interactions [55][56][57][58]. With the advent of numerous protocols for deriving iPSCs from multiple patientderived cell sources, academic and industry partnerships should be used to derive large and comprehensive repositories for iPSCs from well-characterized sibling controls and patients suffering from a range of CNS indications.…”

Section: How Ipscs Can Enhance the Utility Of In Vitro Models Of Diseasementioning

confidence: 99%

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System

Hunsberger

Efthymiou

Malik

et al. 2015

Stem Cells and Development

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There is great need to develop more predictive drug discovery tools to identify new therapies to treat diseases of the central nervous system (CNS). Current nonpluripotent stem cell-based models often utilize non-CNS immortalized cell lines and do not enable the development of personalized models of disease. In this review, we discuss why in vitro models are necessary for translational research and outline the unique advantages of induced pluripotent stem cell (iPSC)-based models over those of current systems. We suggest that iPSC-based models can be patient specific and isogenic lines can be differentiated into many neural cell types for detailed comparisons. iPSC-derived cells can be combined to form small organoids, or large panels of lines can be developed that enable new forms of analysis. iPSC and embryonic stem cell-derived cells can be readily engineered to develop reporters for lineage studies or mechanism of action experiments further extending the utility of iPSC-based systems. We conclude by describing novel technologies that include strategies for the development of diversity panels, novel genomic engineering tools, new three-dimensional organoid systems, and modified high-content screens that may bring toxicology into the 21st century. The strategic integration of these technologies with the advantages of iPSC-derived cell technology, we believe, will be a paradigm shift for toxicology and drug discovery efforts.Disease Modeling D iseases of the central nervous system (CNS) affect a large number of people, but therapeutic intervention is hampered by the lack of useful models for many of these diseases. Current research on human subjects, particularly for drug discovery for CNS diseases, is severely (and appropriately) limited by ethical guidelines. Therefore, surrogate models are needed that share important anatomical, physiological, and genetic features to advance new treatments and therapies for CNS diseases [1].Developing rapid and effective therapies for CNS diseases requires the availability of in vitro models that accurately recapitulate disease phenotypes and predict patient treatment response. A proper model must be both sensitive and predictive while reflecting both normal and disease processes. Equally important, these models should enable the investigation of genetic and environmental risk factors contributing to diseases in a rapid and economical way. Currently used models often do not reflect a typical human response [2-4], despite efforts underway to better characterize these models and increase their preclinical value in predicting safety and efficacy in the clinic [5,6]. Therefore, there is a great need to develop disease-and patient-specific models from cells directly affected in CNS disorders. These cellbased models, we envision, could either replace or supplement current animal models and enable the efficient translation of basic research into the clinical setting. Limitations with current CNS modelsCurrently, drug discovery relies on the use of animalbased or cell-based models, ...

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Rapid and Efficient Generation of Functional Motor Neurons From Human Pluripotent Stem Cells Using Gene Delivered Transcription Factor Codes

Cited by 167 publications

References 50 publications

High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1

High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1

Fusion protein Isl1–Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System

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