Read with caution!

This post was written during early stages of trying to understand a complex scientific problem, and we didn't get everything right. The original author no longer endorses the content of this post. It is being left online for historical reasons, but read at your own risk.

This post is intended to provide a quick, initial look at the state of the field of iPS cells (induced pluripotent stem cells) — who is working with them, and who is working on differentiating them into neurons, and how far along are they.

iPS cells are pluripotent stem cells induced from adult somatic cells. In 2006 Yamanaka et al. published a method for generating iPS cells by exposing adult fibroblasts to four specific growth factors in culture. This is pretty new stuff and has attracted a lot of attention. iPS cells both avoid the ethical issues of embryonic stem cells, and have the added advantage of potentially being patient-specific, circumventing the immune rejection issues faced by ESCs. They have promise for cell replacement therapy, drug screenings, and research into disease mechanisms. That said, the art of figuring out how to control the fates of the cells, and get them to differentiate from their pluripotent state into the cell type of interest, is still a work in progress. And neurons are especially complex and delicate — but also especially interesting. Unlike with many other types of cells taking a biopsy from a live patient in a disease state isn’t an option for neurons. iPS cells may finally provide us with a way to actually see neurodegenerative pathology in cells as it is happening.

Markers and Methods for Cell Sorting of Human Embryonic Stem Cell-Derived Neural Cell Populations

Jan Pruszak,
Kai-Christian Sonntag,
Moe Hein Aung,
Rosario Sanchez-Pernaute,
Ole Isacson M.D.^*

Neuroregeneration Laboratories, Center for Neuroregeneration Research, Udall Parkinson’s Disease Center of Excellence, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA

STEM CELLS, Volume 25, Issue 9, pages 2257–2268, September 2007

http://onlinelibrary.wiley.com./doi/10.1634/stemcells.2006-0744/full

Abstract

Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate better-defined cell populations, we established a working protocol for sorting heterogeneous hESC-derived neural cell populations by fluorescence-activated cell sorting (FACS). Using genetically labeled synapsin-green fluorescent protein-positive hESC-derived neurons as a proof of principle, we enriched viable differentiated neurons by FACS. Cell sorting methodology using surface markers was developed, and a comprehensive profiling of surface antigens was obtained for immature embryonic stem cell types (such as stage-specific embryonic antigen [SSEA]-3, -4, TRA-1-81, TRA-1-60), neural stem and precursor cells (such as CD133, SSEA-1 [CD15], A2B5, forebrain surface embryonic antigen-1, CD29, CD146, p75 [CD271]), and differentiated neurons (such as CD24 or neural cell adhesion molecule [NCAM; CD56]). At later stages of neural differentiation, NCAM (CD56) was used to isolate hESC-derived neurons by FACS. Such FACS-sorted hESC-derived neurons survived in vivo after transplantation into rodent brain. These results and concepts provide (a) a feasible approach for experimental cell sorting of differentiated neurons, (b) an initial survey of surface antigens present during neural differentiation of hESC, and (c) a framework for developing cell selection strategies for neural cell-based therapies.

Induced pluripotent stem cells (iPSCs) and neurological disease modeling: progress and promises

1. Laboratory of Genetics (LOG-G), The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

Hum. Mol. Genet. (2011) 20 (R2): R109-R115.Hum. Mol. Genet. (2011) 20 (R2): R109-R115.

http://hmg.oxfordjournals.org./content/20/R2/R109.full

The systematic generation of neurons from patients with neurological disorders can provide important insights into disease pathology, progression and mechanism. This review will discuss recent progress in modeling neurodegenerative and neurodevelopmental diseases using induced pluripotent stem cells (iPSCs) and highlight some of the current challenges in the field. Combined with other technologies previously used to study brain disease, iPSC modeling has the promise to influence modern medicine on several fronts: early diagnosis, drug development and effective treatment.

Challenges in modeling neurological disease

iPSC technology has a clear potential for identifying the molecular mechanisms of an array of neurological diseases that currently have no cure or effective therapy. Nevertheless, there are a number of pressing issues that need to be addressed before iPSC technology can be extensively used for clinically relevant modeling of neurological diseases. Among these issues are variability in iPSC generation methods, variability between individuals, epigenetic/genetic instability and the ability to obtain disease-relevant subtypes of neurons.

Recently, researchers have begun to assess (and quantify) the variability that is present in iPSC lines. Increased levels of aneuploidy (39), defects in X-chromosome inactivation and genomic imprinting (40,41), aberrant epigenetic reprogramming (42), presence of point mutations and copy number variation differences (43,44) have all been detected in various iPSC lines. It is unclear that which of these differences might be relevant in iPSC disease modeling, as both the expected somatic variability and the level of genetic mosaicism observed within the lifetime of normal individuals remains unknown. Without this knowledge, we cannot yet fully judge the implications of the variability seen between iPSC cultures.

Genomic/epigenomic variability can influence the neuronal differentiation potential of iPSCs (45,46). This variability has been attributed to the use of randomly integrating viral vectors to introduce the reprogramming factors. However, it remains unclear whether novel non-integrating methods will decrease this variability or if the variability instead reflects inherent differences between iPSC lines (47–50). Nonetheless, many published reports have overcome this variance and detected significant phenotypic differences between neuronal cultures from patients with neurological diseases and unaffected controls. Gain- and loss-of-function studies, when possible, can verify that the phenotype observed is specific to the mutated gene and not due to acquired genetic/epigenetic variability.

Current protocols for differentiating iPSC into specific subtypes of neurons are under development. As researchers strive to identify ideal combinations and concentrations of growth factors critical to human neural development, clues can be found in mouse neural embryology studies, though adaptation is required as the developmental timing differs substantially between the two species. Understanding the molecular players involved in human neural differentiation will facilitate the development of methods and tools to enrich and monitor the generation of specific subtypes of neurons that would be more relevant in modeling different neurological diseases. Particularly, promoter bashing techniques could be used to identify different subtypes of neurons for live imaging (51–53), and fluorescent-activated cell sorting using cell surface neuronal markers could be used to purify homogeneous populations (54,55). To date, greater progress has been made in generating enriched populations of ventral midbrain dopaminergic neurons that are relevant for PD (56,57) and spinal motor neurons that are important players during ALS pathology (13). Some progress has been made in regionalizing human ESCs into forebrain cholinergic neurons, often affected in AD, but iPSCs have not yet been subjected to these protocols (10,58).

Recapitulation of human corticogenesis in vitro has also been a challenge (59). Modeling for diseases where organization of cortical layers is proposed to be altered, such as Autism (60) and Schizophrenia (61), would benefit from protocols that accurately generate enriched populations of cortical neurons. Compartmentalization and stratification of neurons using chamber devices associated with live imaging would be useful to start teasing out the dynamic behavior and molecular anatomy of those neurons in a more refined way (62,63).

Recently, several groups have reported the direct conversion of somatic cells to post-mitotic neurons, skipping an iPSC intermediate (64–67). While promising, this technology could be limited by the subtypes of neurons generated, decreased efficiency and the finite proliferative capabilities of most somatic cell sources. Primary cells typically senesce after consecutive passaging, whereas iPSCs have nearly limitless replicative abilities.

Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

Yuan SH, Martin J, Elia J, Flippin J, Paramban RI, et al. (2011) PLoS ONE 6(3): e17540.

1 Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 3 BD Biosciences, La Jolla, California, United States of America, 4 Anesthesiology Research Laboratory, Department of Anesthesiology, University of California San Diego, La Jolla, California, United States of America, 5 Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California, United States of America, 6 Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California, United States of America, 7 Institute of Neurobiology, Slovak Academy of Sciences, Košice, Slovakia

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0017540

Background

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).

Methodology/Principal Findings

We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184⁺/CD271⁻/CD44⁻/CD24⁺ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184⁻/CD44⁻/CD15^LOW/CD24⁺ and a population of glia that was CD184⁺/CD44⁺ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.

Conclusions/ Significance

These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

Direct conversion of fibroblasts to functional neurons by defined factors

Nature 463, 1035-1041 (25 February 2010) | doi:10.1038/nature08797; Received 9 October 2009; Accepted 6 January 2010; Published online 27 January 2010

Thomas Vierbuchen¹^,², Austin Ostermeier¹^,², Zhiping P. Pang³, Yuko Kokubu¹, Thomas C. Südhof³^,⁴ & Marius Wernig¹^,²

Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology,
Program in Cancer Biology,

3. Department of Molecular and Cellular Physiology,

4. Howard Hughes Medical Institute, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, California 94304, USA

http://www.nature.com./nature/journal/v463/n7284/full/nature08797.html

Abstract

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

**Functional Properties of Neurons Derived from In Vitro Reprogrammed Postnatal Astroglia**

1. ¹Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany,

2. ²Institute for Stem Cell Research, National Research Center for Environment and Health, D-85764 Neuherberg, Germany, and

3. ³Department Biologie II, Division of Neurobiology, Ludwig-Maximilians University Munich, D-82152 Planegg-Martinsried, Germany

http://www.jneurosci.org./content/27/32/8654.full

Abstract

With the exception of astroglia-like cells in the neurogenic niches of the telencephalic subependymal or hippocampal subgranular zone, astroglia in all other regions of the adult mouse brain do not normally generate neurons. Previous studies have shown, however, that early postnatal cortical astroglia in culture can be reprogrammed to adopt a neuronal fate after forced expression of Pax6, a transcription factor (TF) required for proper neuronal specification during embryonic corticogenesis. Here we show that also the proneural genes neurogenin-2 and Mash1 (mammalian achaete schute homolog 1) possess the ability to reprogram astroglial cells from early postnatal cerebral cortex. By means of time-lapse imaging of green fluorescent astroglia, we provide direct evidence that it is indeed cells with astroglial characteristics that give rise to neurons. Using patch-clamp recordings in culture, we show that astroglia-derived neurons acquire active conductances and are capable of firing action potentials, thus displaying hallmarks of true neurons. However, independent of the TF used for reprogramming, astroglia-derived neurons appear to mature more slowly compared with embryonic-born neurons and fail to generate a functional presynaptic output within the culturing period. However, when cocultured with embryonic cortical neurons, astroglia-derived neurons receive synaptic input, demonstrating that they are competent of establishing a functional postsynaptic compartment. Our data demonstrate that single TFs are capable of inducing a remarkable functional reprogramming of astroglia toward a truly neuronal identity.

Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency

1. ^aWaisman Center,

2. ^bGenome Center,

3. ^cDepartment of Anatomy, and

4. ^dDepartment of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705

1. Edited by Rudolf Jaenisch, Whitehead Institute for Biomedical Research, Cambridge, MA, and approved January 19, 2010 (received for review September 1, 2009)

http://www.pnas.org./content/107/9/4335.abstract?ijkey=0aac16663b5aecdf4681e6ff33ead364b42fadb7&keytype2=tf_ipsecsha

Abstract

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Nature Biotechnology 27, 275 – 280 (2009)
Published online: 1 March 2009 | Corrected online: 16 March 2009 | doi:10.1038/nbt.1529

Stuart M Chambers¹, Christopher A Fasano¹, Eirini P Papapetrou², Mark Tomishima¹^,², Michel Sadelain²^,³ & Lorenz Studer¹^,²^,⁴

Developmental Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA.
Center for Cell Engineering, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA.
Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA
Department of Neurosurgery, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA.

http://www.nature.com./nbt/journal/v27/n3/full/nbt.1529.html

Abstract

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5⁺ epiblast-like stage followed by PAX6⁺ neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

Direct conversion of human fibroblasts to dopaminergic neurons

1. Departments of Experimental Medical Science and Clinical Sciences, Wallenberg Neuroscience Center, and Lund Stem Cell Center, Lund University, SE-221 84 Lund, Sweden

1. Edited* by Fred H. Gage, The Salk Institute, San Diego, CA, and approved May 13, 2011 (received for review March 31, 2011)

http://www.pnas.org./content/108/25/10343.abstract?ijkey=1ca1385b3483ccfb9938f5bff43d4b546caeb08b&keytype2=tf_ipsecsha

Abstract

Recent reports demonstrate that somatic mouse cells can be directly converted to other mature cell types by using combined expression of defined factors. Here we show that the same strategy can be applied to human embryonic and postnatal fibroblasts. By overexpression of the transcription factors Ascl1, Brn2, and Myt1l, human fibroblasts were efficiently converted to functional neurons. We also demonstrate that the converted neurons can be directed toward distinct functional neurotransmitter phenotypes when the appropriate transcriptional cues are provided together with the three conversion factors. By combining expression of the three conversion factors with expression of two genes involved in dopamine neuron generation, Lmx1a and FoxA2, we could direct the phenotype of the converted cells toward dopaminergic neurons. Such subtype-specific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy.

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

Karl R Koehler¹^,², Philippe Tropel⁵, Jonathan W Theile³^,¹, Takako Kondo¹^,², Theodore R Cummins³^,¹, Stéphane Viville⁴^,⁵ and Eri Hashino³^,¹^,²^*

¹ Stark Neurosciences Research Institute

² Department of Otolaryngology

³ Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

⁴ Service de Biologie de la Reproduction, Centre Hospitalier Universitaire, Strasbourg, F-67000 France

⁵ Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 1704/Université de Strasbourg, 67404 Illkirch, France

BMC Neuroscience 2011, 12:82 doi:10.1186/1471-2202-12-82

http://www.biomedcentral.com/1471-2202/12/82

The use of induced pluripotent stem cells (iPSCs) for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC) neural induction protocol to test whether iPSCs (1) have the competence to give rise to functional neurons with similar efficiency as ESCs and (2) whether the extent of neural differentiation could be altered or enhanced by increased passaging.

Results

Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs) expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs). Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable.

Conclusions

These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease

1. *The Whitehead Institute for Biomedical Research, Cambridge, MA 02142;

2. ^†The McGovern Institute for Brain Research and

3. ^¶Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;

4. ^‡Udall Parkinson’s Disease Research Center of Excellence and Neuroregeneration Laboratories, McLean Hospital/Harvard University, Belmont, MA 02478; and

5. ^§San Raffaele Scientific Institute, 20132 Milan, Italy

PNAS April 15, 2008 vol. 105 no. 15 5856-5861

http://www.pnas.org./content/105/15/5856.full

Abstract

The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson’s disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.

Markers and Methods for Cell Sorting Human Embryonic Stem Cell-Derived Neural Cell Populations

Jan Pruszak,
Kai-Christian Sonntag,
Moe Hein Aung,
Rosario Sanchez-Pernaute,
Ole Isacson M.D.^*

Neuroregeneration Laboratories, Center for Neuroregeneration Research, Udall Parkinson’s Disease Center of Excellence, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA

STEM CELLS, Volume 25, Issue 9, pages 2257–2268, September 2007

http://onlinelibrary.wiley.com./doi/10.1634/stemcells.2006-0744/full

Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate better-defined cell populations, we established a working protocol for sorting heterogeneous hESC-derived neural cell populations by fluorescence-activated cell sorting (FACS). Using genetically labeled synapsin-green fluorescent protein-positive hESC-derived neurons as a proof of principle, we enriched viable differentiated neurons by FACS. Cell sorting methodology using surface markers was developed, and a comprehensive profiling of surface antigens was obtained for immature embryonic stem cell types (such as stage-specific embryonic antigen [SSEA]-3, -4, TRA-1-81, TRA-1-60), neural stem and precursor cells (such as CD133, SSEA-1 [CD15], A2B5, forebrain surface embryonic antigen-1, CD29, CD146, p75 [CD271]), and differentiated neurons (such as CD24 or neural cell adhesion molecule [NCAM; CD56]). At later stages of neural differentiation, NCAM (CD56) was used to isolate hESC-derived neurons by FACS. Such FACS-sorted hESC-derived neurons survived in vivo after transplantation into rodent brain. These results and concepts provide (a) a feasible approach for experimental cell sorting of differentiated neurons, (b) an initial survey of surface antigens present during neural differentiation of hESC, and (c) a framework for developing cell selection strategies for neural cell-based therapies.

Induced pluripotent stem cells (iPSCs) and neurological disease modeling: progress and promises

1. Laboratory of Genetics (LOG-G), The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA

Hum. Mol. Genet. (2011) 20 (R2): R109-R115.Hum. Mol. Genet. (2011) 20 (R2): R109-R115.

http://hmg.oxfordjournals.org./content/20/R2/R109.full

The systematic generation of neurons from patients with neurological disorders can provide important insights into disease pathology, progression and mechanism. This review will discuss recent progress in modeling neurodegenerative and neurodevelopmental diseases using induced pluripotent stem cells (iPSCs) and highlight some of the current challenges in the field. Combined with other technologies previously used to study brain disease, iPSC modeling has the promise to influence modern medicine on several fronts: early diagnosis, drug development and effective treatment.

Challenges in modeling neurological disease

iPSC technology has a clear potential for identifying the molecular mechanisms of an array of neurological diseases that currently have no cure or effective therapy. Nevertheless, there are a number of pressing issues that need to be addressed before iPSC technology can be extensively used for clinically relevant modeling of neurological diseases. Among these issues are variability in iPSC generation methods, variability between individuals, epigenetic/genetic instability and the ability to obtain disease-relevant subtypes of neurons.

Recently, researchers have begun to assess (and quantify) the variability that is present in iPSC lines. Increased levels of aneuploidy (39), defects in X-chromosome inactivation and genomic imprinting (40,41), aberrant epigenetic reprogramming (42), presence of point mutations and copy number variation differences (43,44) have all been detected in various iPSC lines. It is unclear that which of these differences might be relevant in iPSC disease modeling, as both the expected somatic variability and the level of genetic mosaicism observed within the lifetime of normal individuals remains unknown. Without this knowledge, we cannot yet fully judge the implications of the variability seen between iPSC cultures.

Genomic/epigenomic variability can influence the neuronal differentiation potential of iPSCs (45,46). This variability has been attributed to the use of randomly integrating viral vectors to introduce the reprogramming factors. However, it remains unclear whether novel non-integrating methods will decrease this variability or if the variability instead reflects inherent differences between iPSC lines (47–50). Nonetheless, many published reports have overcome this variance and detected significant phenotypic differences between neuronal cultures from patients with neurological diseases and unaffected controls. Gain- and loss-of-function studies, when possible, can verify that the phenotype observed is specific to the mutated gene and not due to acquired genetic/epigenetic variability.

Current protocols for differentiating iPSC into specific subtypes of neurons are under development. As researchers strive to identify ideal combinations and concentrations of growth factors critical to human neural development, clues can be found in mouse neural embryology studies, though adaptation is required as the developmental timing differs substantially between the two species. Understanding the molecular players involved in human neural differentiation will facilitate the development of methods and tools to enrich and monitor the generation of specific subtypes of neurons that would be more relevant in modeling different neurological diseases. Particularly, promoter bashing techniques could be used to identify different subtypes of neurons for live imaging (51–53), and fluorescent-activated cell sorting using cell surface neuronal markers could be used to purify homogeneous populations (54,55). To date, greater progress has been made in generating enriched populations of ventral midbrain dopaminergic neurons that are relevant for PD (56,57) and spinal motor neurons that are important players during ALS pathology (13). Some progress has been made in regionalizing human ESCs into forebrain cholinergic neurons, often affected in AD, but iPSCs have not yet been subjected to these protocols (10,58).

Recapitulation of human corticogenesis in vitro has also been a challenge (59). Modeling for diseases where organization of cortical layers is proposed to be altered, such as Autism (60) and Schizophrenia (61), would benefit from protocols that accurately generate enriched populations of cortical neurons. Compartmentalization and stratification of neurons using chamber devices associated with live imaging would be useful to start teasing out the dynamic behavior and molecular anatomy of those neurons in a more refined way (62,63).

Recently, several groups have reported the direct conversion of somatic cells to post-mitotic neurons, skipping an iPSC intermediate (64–67). While promising, this technology could be limited by the subtypes of neurons generated, decreased efficiency and the finite proliferative capabilities of most somatic cell sources. Primary cells typically senesce after consecutive passaging, whereas iPSCs have nearly limitless replicative abilities.

Cell-surface marker signatures for the Isolation of Neuron Stem cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

Yuan SH, Martin J, Elia J, Flippin J, Paramban RI, et al. (2011) PLoS ONE 6(3): e17540.

doi:10.1371/journal.pone.0017540

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0017540

Background

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).

Methodology/ Principal Findings

We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184⁺/CD271⁻/CD44⁻/CD24⁺ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184⁻/CD44⁻/CD15^LOW/CD24⁺ and a population of glia that was CD184⁺/CD44⁺ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.

Conclusions/ Significance

These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

Direct conversion of fibroblasts to functional neurons by defined factors

Nature 463, 1035-1041 (25 February 2010) | doi:10.1038/nature08797; Received 9 October 2009; Accepted 6 January 2010; Published online 27 January 2010

Thomas Vierbuchen¹^,², Austin Ostermeier¹^,², Zhiping P. Pang³, Yuko Kokubu¹, Thomas C. Südhof³^,⁴ & Marius Wernig¹^,²

Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology,
Program in Cancer Biology,

3. Department of Molecular and Cellular Physiology,

4. Howard Hughes Medical Institute, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, California 94304, USA

http://www.nature.com./nature/journal/v463/n7284/full/nature08797.html

Abstract

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

Functional Properties of Neurons Derived from In Vitro Reprogrammed Postnatal Astroglia

1. ¹Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, D-80336 Munich, Germany,

2. ²Institute for Stem Cell Research, National Research Center for Environment and Health, D-85764 Neuherberg, Germany, and

3. ³Department Biologie II, Division of Neurobiology, Ludwig-Maximilians University Munich, D-82152 Planegg-Martinsried, Germany

http://www.jneurosci.org./content/27/32/8654.full

Abstract

With the exception of astroglia-like cells in the neurogenic niches of the telencephalic subependymal or hippocampal subgranular zone, astroglia in all other regions of the adult mouse brain do not normally generate neurons. Previous studies have shown, however, that early postnatal cortical astroglia in culture can be reprogrammed to adopt a neuronal fate after forced expression of Pax6, a transcription factor (TF) required for proper neuronal specification during embryonic corticogenesis. Here we show that also the proneural genes neurogenin-2 and Mash1 (mammalian achaete schute homolog 1) possess the ability to reprogram astroglial cells from early postnatal cerebral cortex. By means of time-lapse imaging of green fluorescent astroglia, we provide direct evidence that it is indeed cells with astroglial characteristics that give rise to neurons. Using patch-clamp recordings in culture, we show that astroglia-derived neurons acquire active conductances and are capable of firing action potentials, thus displaying hallmarks of true neurons. However, independent of the TF used for reprogramming, astroglia-derived neurons appear to mature more slowly compared with embryonic-born neurons and fail to generate a functional presynaptic output within the culturing period. However, when cocultured with embryonic cortical neurons, astroglia-derived neurons receive synaptic input, demonstrating that they are competent of establishing a functional postsynaptic compartment. Our data demonstrate that single TFs are capable of inducing a remarkable functional reprogramming of astroglia toward a truly neuronal identity.

Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency

1. ^aWaisman Center,

2. ^bGenome Center,

3. ^cDepartment of Anatomy, and

4. ^dDepartment of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705

1. Edited by Rudolf Jaenisch, Whitehead Institute for Biomedical Research, Cambridge, MA, and approved January 19, 2010 (received for review September 1, 2009)

http://www.pnas.org./content/107/9/4335.abstract?ijkey=0aac16663b5aecdf4681e6ff33ead364b42fadb7&keytype2=tf_ipsecsha

For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Nature Biotechnology 27, 275 – 280 (2009)
Published online: 1 March 2009 | Corrected online: 16 March 2009 | doi:10.1038/nbt.1529

Stuart M Chambers¹, Christopher A Fasano¹, Eirini P Papapetrou², Mark Tomishima¹^,², Michel Sadelain²^,³ & Lorenz Studer¹^,²^,⁴

Developmental Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA.
Center for Cell Engineering, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA.
Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA
Department of Neurosurgery, Sloan-Kettering Institute, 1275 York Ave., New York, New York 10065, USA.

http://www.nature.com./nbt/journal/v27/n3/full/nbt.1529.html

Abstract

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5⁺ epiblast-like stage followed by PAX6⁺ neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

Direct conversion of human fibroblasts to dopaminergic neurons

1. Departments of Experimental Medical Science and Clinical Sciences, Wallenberg Neuroscience Center, and Lund Stem Cell Center, Lund University, SE-221 84 Lund, Sweden

1. Edited* by Fred H. Gage, The Salk Institute, San Diego, CA, and approved May 13, 2011 (received for review March 31, 2011)

http://www.pnas.org./content/108/25/10343.abstract?ijkey=1ca1385b3483ccfb9938f5bff43d4b546caeb08b&keytype2=tf_ipsecsha

Recent reports demonstrate that somatic mouse cells can be directly converted to other mature cell types by using combined expression of defined factors. Here we show that the same strategy can be applied to human embryonic and postnatal fibroblasts. By overexpression of the transcription factors Ascl1, Brn2, and Myt1l, human fibroblasts were efficiently converted to functional neurons. We also demonstrate that the converted neurons can be directed toward distinct functional neurotransmitter phenotypes when the appropriate transcriptional cues are provided together with the three conversion factors. By combining expression of the three conversion factors with expression of two genes involved in dopamine neuron generation, Lmx1a and FoxA2, we could direct the phenotype of the converted cells toward dopaminergic neurons. Such subtype-specific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy.

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

Karl R Koehler¹^,², Philippe Tropel⁵, Jonathan W Theile³^,¹, Takako Kondo¹^,², Theodore R Cummins³^,¹, Stéphane Viville⁴^,⁵ and Eri Hashino³^,¹^,²^*

¹ Stark Neurosciences Research Institute

² Department of Otolaryngology

³ Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

⁴ Service de Biologie de la Reproduction, Centre Hospitalier Universitaire, Strasbourg, F-67000 France

BMC Neuroscience 2011, 12:82 doi:10.1186/1471-2202-12-82

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2202/12/82

Published:	10 August 2011

Background

The use of induced pluripotent stem cells (iPSCs) for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC) neural induction protocol to test whether iPSCs (1) have the competence to give rise to functional neurons with similar efficiency as ESCs and (2) whether the extent of neural differentiation could be altered or enhanced by increased passaging.

Results

Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs) expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs). Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable.

Conclusions

These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease

10. *The Whitehead Institute for Biomedical Research, Cambridge, MA 02142;

11. ^†The McGovern Institute for Brain Research and

12. ^¶Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;

13. ^‡Udall Parkinson’s Disease Research Center of Excellence and Neuroregeneration Laboratories, McLean Hospital/Harvard University, Belmont, MA 02478; and

14. ^§San Raffaele Scientific Institute, 20132 Milan, Italy

Rudolf Jaenisch

PNAS April 15, 2008 vol. 105 no. 15 5856-5861

http://www.pnas.org./content/105/15/5856.full

The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson’s disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.