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1.  Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells 
BMC Neuroscience  2011;12:82.
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.
doi:10.1186/1471-2202-12-82
PMCID: PMC3167757  PMID: 21831300
2.  Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells 
Introduction
Human induced pluripotent stem cells (hiPSCs) offer great promise for regenerative therapies or in vitro modelling of neurodegenerative disorders like Parkinson’s disease. Currently, widely used cell sources for the generation of hiPSCs are somatic cells obtained from aged individuals. However, a critical issue concerning the potential clinical use of these iPSCs is mutations that accumulate over lifetime and are transferred onto iPSCs during reprogramming which may influence the functionality of cells differentiated from them. The aim of our study was to establish a differentiation strategy to efficiently generate neurons including dopaminergic cells from human cord blood-derived iPSCs (hCBiPSCs) as a juvenescent cell source and prove their functional maturation in vitro.
Methods
The differentiation of hCBiPSCs was initiated by inhibition of transforming growth factor-β and bone morphogenetic protein signaling using the small molecules dorsomorphin and SB 431542 before final maturation was carried out. hCBiPSCs and differentiated neurons were characterized by immunocytochemistry and quantitative real time-polymerase chain reaction. Since functional investigations of hCBiPSC-derived neurons are indispensable prior to clinical applications, we performed detailed analysis of essential ion channel properties using whole-cell patch-clamp recordings and calcium imaging.
Results
A Sox1 and Pax6 positive neuronal progenitor cell population was efficiently induced from hCBiPSCs using a newly established differentiation protocol. Neuronal progenitor cells could be further maturated into dopaminergic neurons expressing tyrosine hydroxylase, the dopamine transporter and engrailed 1. Differentiated hCBiPSCs exhibited voltage-gated ion currents, were able to fire action potentials and displayed synaptic activity indicating synapse formation. Application of the neurotransmitters GABA, glutamate and acetylcholine induced depolarizing calcium signal changes in neuronal cells providing evidence for the excitatory effects of these ligand-gated ion channels during maturation in vitro.
Conclusions
This study demonstrates for the first time that hCBiPSCs can be used as a juvenescent cell source to generate a large number of functional neurons including dopaminergic cells which may serve for the development of novel regenerative treatment strategies.
doi:10.1186/scrt423
PMCID: PMC4055096  PMID: 24636737
3.  Generation of Mice Derived from Induced Pluripotent Stem Cells 
The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.
doi:10.3791/4003
PMCID: PMC3565852  PMID: 23222420
Stem Cell Biology; Issue 69; Molecular Biology; Developmental Biology; Medicine; Cellular Biology; Induced pluripotent stem cells; iPSC; stem cells; reprogramming; developmental potential; tetraploid embryo complementation; mouse
4.  Efficient Generation of Functional Dopaminergic Neurons from Human Induced Pluripotent Stem Cells Under Defined Conditions 
Stem Cells (Dayton, Ohio)  2010;28(10):1893-1904.
Human induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells represent a promising unlimited cell source for generating patient-specific cells for biomedical research and personalized medicine. As a first step, critical to clinical applications, we attempted to develop defined culture conditions to expand and differentiate human iPSCs into functional progeny such as dopaminergic neurons for treating or modeling Parkinson's disease (PD). We used a completely defined (xeno-free) system that we previously developed for efficient generation of authentic dopaminergic neurons from human embryonic stem cells (hESCs), and applied it to iPSCs. First, we adapted two human iPSC lines derived from different somatic cell types for the defined expansion medium and showed that the iPSCs grew similarly as hESCs in the same medium regarding pluripotency and genomic stability. Second, by using these two independent adapted iPSC lines, we showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was also similar to hESCs. Importantly, iPSC-derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. In addition, iPSC-derived NSCs and neurons could be efficiently transduced by a baculoviral vector delivering episomal DNA for future gene function study and disease modeling using iPSCs. We also performed genome-wide microarray comparisons between iPSCs and hESCs, and we derived NSC and dopaminergic neurons. Our data revealed overall similarity and visible differences at a molecular level. Efficient generation of functional dopaminergic neurons under defined conditions will facilitate research and applications using PD patient-specific iPSCs. Stem Cells 2010;28:1893–1904
doi:10.1002/stem.499
PMCID: PMC2996088  PMID: 20715183
Human iPSC; Human ESC; Dopaminergic neuron; Neural stem cell
5.  Differentiation of spinal motor neurons from pluripotent human stem cells 
Nature protocols  2009;4(9):1295-1304.
We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post- mitotic, HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8–10 weeks for the production of functional mature motoneurons. In comparison with other methods, our protocol does not use feeder cells, has a minimum dependence on proteins (purmorphamine replacing SHH), has controllable adherent selection and is adaptable for scalable suspension culture.
doi:10.1038/nprot.2009.127
PMCID: PMC2789120  PMID: 19696748
6.  A Novel Model of Urinary Tract Differentiation, Tissue Regeneration, and Disease: Reprogramming Human Prostate and Bladder Cells into Induced Pluripotent Stem Cells 
European Urology  2013;64(5):753-761.
Background
Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.
Objective
To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.
Design, setting, and participants
Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.
Outcome measurements and statistical analysis
The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.
Results and limitations
Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.
Conclusions
IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.
Take Home Message
Induced pluripotent stem cells derived from human prostate and urinary tract cells can differentiate back into prostate and bladder cells. These cells provide a convenient ready-to-access model that offers enormous potential in clinical regenerative medicine and disease modelling.
doi:10.1016/j.eururo.2013.03.054
PMCID: PMC3819995  PMID: 23582880
Prostate; Bladder; Differentiation; Pluripotent; Stem cells; Tissue engineering; Ureter; Urothelium; Androgen receptor; POU5F1 (formerly OCT4); SOX2; KLF4; MYC (formerly cMYC); NANOG
7.  Dual Small-Molecule Targeting of SMAD Signaling Stimulates Human Induced Pluripotent Stem Cells toward Neural Lineages 
PLoS ONE  2014;9(9):e106952.
Incurable neurological disorders such as Parkinson’s disease (PD), Huntington’s disease (HD), and Alzheimer’s disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases, we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor, valproic acid (VPA), and inhibitor of p160-Rho associated coiled-coil kinase (ROCK), Y-27632, after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology, cell surface antigens, pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542, inhibitors of the SMAD signaling pathway, HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction, neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture, which had the ability to differentiate further into NPCs and neurons, as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays.
doi:10.1371/journal.pone.0106952
PMCID: PMC4160199  PMID: 25207966
8.  Transcripts involved in calcium signaling and telencephalic neuronal fate are altered in induced pluripotent stem cells from bipolar disorder patients 
Translational Psychiatry  2014;4(3):e375-.
Bipolar disorder (BP) is a chronic psychiatric condition characterized by dynamic, pathological mood fluctuations from mania to depression. To date, a major challenge in studying human neuropsychiatric conditions such as BP has been limited access to viable central nervous system tissue to examine disease progression. Patient-derived induced pluripotent stem cells (iPSCs) now offer an opportunity to analyze the full compliment of neural tissues and the prospect of identifying novel disease mechanisms. We have examined changes in gene expression as iPSC derived from well-characterized patients differentiate into neurons; there was little difference in the transcriptome of iPSC, but BP neurons were significantly different than controls in their transcriptional profile. Expression of transcripts for membrane bound receptors and ion channels was significantly increased in BP-derived neurons compared with controls, and we found that lithium pretreatment of BP neurons significantly altered their calcium transient and wave amplitude. The expression of transcription factors involved in the specification of telencephalic neuronal identity was also altered. Control neurons expressed transcripts that confer dorsal telencephalic fate, whereas BP neurons expressed genes involved in the differentiation of ventral (medial ganglionic eminence) regions. Cells were responsive to dorsal/ventral patterning cues, as addition of the Hedgehog (ventral) pathway activator purmorphamine or a dorsalizing agent (lithium) stimulated expression of NKX2-1 (ventral identity) or EMX2 (dorsal) in both groups. Cell-based models should have a significant impact on our understanding of the genesis and therefore treatment of BP; the iPSC cell lines themselves provide an important resource for comparison with other neurodevelopmental disorders.
doi:10.1038/tp.2014.12
PMCID: PMC3966040  PMID: 25116795
calcium signaling; disease modeling; microarray analysis; neuronal differentiation
9.  Reprogramming of Fibroblasts From Older Women With Pelvic Floor Disorders Alters Cellular Behavior Associated With Donor Age 
The effect of donor age on induced pluripotent stem cell (iPSC) lines and on the cells redifferentiated from these iPSCs was examined. iPSCs were derived from vaginal fibroblasts from women with pelvic organ prolapse. Donor age did not appear to affect reprogramming and cell mitotic activity in fibroblasts redifferentiated from iPSCs, and donor age differences were not observed in the iPSCs using standard senescence markers.
We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated β-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs.
doi:10.5966/sctm.2012-0092
PMCID: PMC3659753  PMID: 23341439
p53; Induced pluripotent stem cells; Reprogramming
10.  Efficient Generation of A9 Midbrain Dopaminergic Neurons by Lentiviral Delivery of LMX1A in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells 
Human Gene Therapy  2011;23(1):56-69.
Abstract
Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) offer great hope for in vitro modeling of Parkinson's disease (PD), as well as for designing cell-replacement therapies. To realize these opportunities, there is an urgent need to develop efficient protocols for the directed differentiation of hESC/iPSC into dopamine (DA) neurons with the specific characteristics of the cell population lost to PD, i.e., A9-subtype ventral midbrain DA neurons. Here we use lentiviral vectors to drive the expression of LMX1A, which encodes a transcription factor critical for ventral midbrain identity, specifically in neural progenitor cells. We show that clonal lines of hESC engineered to contain one or two copies of this lentiviral vector retain long-term self-renewing ability and pluripotent differentiation capacity. Greater than 60% of all neurons generated from LMX1A-engineered hESC were ventral midbrain DA neurons of the A9 subtype, compared with ∼10% in green fluorescent protein–engineered controls, as judged by specific marker expression and functional analyses. Moreover, DA neuron precursors differentiated from LMX1A-engineered hESC were able to survive and differentiate when grafted into the brain of adult mice. Finally, we provide evidence that LMX1A overexpression similarly increases the yield of DA neuron differentiation from human iPSC. Taken together, our data show that stable genetic engineering of hESC/iPSC with lentiviral vectors driving controlled expression of LMX1A is an efficient way to generate enriched populations of human A9-subtype ventral midbrain DA neurons, which should prove useful for modeling PD and may be helpful for designing future cell-replacement strategies.
Sánchez-Danés and colleagues describe a system wherein they use lentiviral vectors to drive the expression of LMX1A, a transcription factor critical for neural progenitor cells, human embryonic stem cells, and induced pluripotent stem cells. They show that this approach can result in the generation of human A9-subtype ventral midbrain dopaminergic neurons, which should prove useful for modeling Parkinson's disease and may be helpful for designing future cell-replacement strategies.
doi:10.1089/hum.2011.054
PMCID: PMC3472681  PMID: 21877920
11.  Genome Wide Profiling of Dopaminergic Neurons Derived from Human Embryonic and Induced Pluripotent Stem Cells 
Stem Cells and Development  2013;23(4):406-420.
Recent advances in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) biology enable generation of dopaminergic neurons for potential therapy and drug screening. However, our current understanding of molecular and cellular signaling that controls human dopaminergic development and function is limited. Here, we report on a whole genome analysis of gene expression during dopaminergic differentiation of human ESC/iPSC using Illumina bead microarrays. We generated a transcriptome data set containing the expression levels of 28,688 unique transcripts by profiling five lines (three ESC and two iPSC lines) at four stages of differentiation: (1) undifferentiated ESC/iPSC, (2) neural stem cells, (3) dopaminergic precursors, and (4) dopaminergic neurons. This data set provides comprehensive information about genes expressed at each stage of differentiation. Our data indicate that distinct pathways are activated during neural and dopaminergic neuronal differentiation. For example, WNT, sonic hedgehog (SHH), and cAMP signaling pathways were found over-represented in dopaminergic populations by gene enrichment and pathway analysis, and their role was confirmed by perturbation analyses using RNAi (small interfering RNA of SHH and WNT) or small molecule [dibutyryl cyclic AMP (dcAMP)]. In summary, whole genome profiling of dopaminergic differentiation enables systematic analysis of genes/pathways, networks, and cellular/molecular processes that control cell fate decisions. Such analyses will serve as the foundation for better understanding of dopaminergic development, function, and development of future stem cell-based therapies.
doi:10.1089/scd.2013.0412
PMCID: PMC3920839  PMID: 24074155
12.  Use of a Synthetic Xeno-Free Culture Substrate for Induced Pluripotent Stem Cell Induction and Retinal Differentiation 
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). It was found that Synthemax cell culture surfaces provide an ideal surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). iPSCs were generated using adult somatic cells via infection with either a single cre-excisable lentiviral vector or four separate nonintegrating Sendai viruses driving expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk-1, noggin, insulin-like growth factor-1, basic fibroblast growth factor, acidic fibroblast growth factor, and DAPT. Phase contrast microscopy, immunocytochemistry, hematoxylin and eosin staining, and reverse transcription-polymerase chain reaction were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs. Following viral transduction, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough for manual isolation/passage at 3–4 weeks postinfection. Both normal and disease-specific iPSCs expressed markers of pluripotency and, following transplantation into immune-compromised mice, formed teratomas containing tissue comprising all three germ layers. When subjected to our established retinal differentiation protocol, a significant proportion of the xeno-free substrate-derived cells expressed retinal cell markers, the number of which did not significantly differ from that derived on traditional extracellular matrix-coated dishes. Synthetic cell culture substrates provide a useful surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
doi:10.5966/sctm.2012-0040
PMCID: PMC3659741  PMID: 23283489
Induced pluripotent stem cells; Stem cell culture; Retina; Reprogramming; Pluripotent stem cells; Clinical translation
13.  Establishment of transgene-free induced pluripotent stem cells reprogrammed from human stem cells of apical papilla for neural differentiation 
Introduction
Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential.
Methods
A single lentiviral 'stem cell cassette' flanked by the loxP site (hSTEMCCA-loxP), encoding four human reprogramming factors, OCT4, SOX2, KLF4, and c-MYC, was used to reprogram human SCAP into iPSCs. Generated iPSCs were transfected with plasmid pHAGE2-EF1α-Cre-IRES-PuroR and selected with puromycin for the TF-iPSC subclones. PCR was performed to confirm the excision of hSTEMCCA. TF-iPSC clones did not resist to puromycin treatment indicating no pHAGE2-EF1α-Cre-IRES-PuroR integration into the genome. In vitro and in vivo analyses of their pluripotency were performed. Embryoid body-mediated neural differentiation was undertaken to verify their neurogenic potential.
Results
TF-SCAP iPSCs were generated via a hSTEMCCA-loxP/Cre system. PCR of genomic DNA confirmed transgene excision and puromycin treatment verified the lack of pHAGE2-EF1α-Cre-IRES-PuroR integration. Transplantation of the TF-iPSCs into immunodeficient mice gave rise to teratomas containing tissues representing the three germ layers -- ectoderm (neural rosettes), mesoderm (cartilage and bone tissues) and endoderm (glandular epithelial tissues). Embryonic stem cell-associated markers TRA-1-60, TRA-2-49 and OCT4 remained positive after transgene excision. After neurogenic differentiation, cells showed neural-like morphology expressing neural markers nestin, βIII-tubulin, NFM, NSE, NeuN, GRM1, NR1 and CNPase.
Conclusions
TF-SCAP iPSCs reprogrammed from SCAP can be generated and they may be a good cell source for neurogenesis.
doi:10.1186/scrt134
PMCID: PMC3580434  PMID: 23095454
14.  Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells 
The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.
doi:10.5966/sctm.2011-0022
PMCID: PMC3659681  PMID: 23197756
Mesenchymal stem cells; Pluripotent stem cells; Differentiation; Induced pluripotent stem cells; Embryonic stem cells
15.  Efficient and Rapid Derivation of Primitive Neural Stem Cells and Generation of Brain Subtype Neurons From Human Pluripotent Stem Cells 
Stem Cells Translational Medicine  2013;2(11):862-870.
This study developed a highly efficient serum-free pluripotent stem cell (PSC) neural induction medium that can induce human PSCs into primitive neural stem cells (NSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. This method of primitive NSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells, are unique cell sources for disease modeling, drug discovery screens, and cell therapy applications. The first step in producing neural lineages from hPSCs is the generation of neural stem cells (NSCs). Current methods of NSC derivation involve the time-consuming, labor-intensive steps of an embryoid body generation or coculture with stromal cell lines that result in low-efficiency derivation of NSCs. In this study, we report a highly efficient serum-free pluripotent stem cell neural induction medium that can induce hPSCs into primitive NSCs (pNSCs) in 7 days, obviating the need for time-consuming, laborious embryoid body generation or rosette picking. The pNSCs expressed the neural stem cell markers Pax6, Sox1, Sox2, and Nestin; were negative for Oct4; could be expanded for multiple passages; and could be differentiated into neurons, astrocytes, and oligodendrocytes, in addition to the brain region-specific neuronal subtypes GABAergic, dopaminergic, and motor neurons. Global gene expression of the transcripts of pNSCs was comparable to that of rosette-derived and human fetal-derived NSCs. This work demonstrates an efficient method to generate expandable pNSCs, which can be further differentiated into central nervous system neurons and glia with temporal, spatial, and positional cues of brain regional heterogeneity. This method of pNSC derivation sets the stage for the scalable production of clinically relevant neural cells for cell therapy applications in good manufacturing practice conditions.
doi:10.5966/sctm.2013-0080
PMCID: PMC3808201  PMID: 24113065
Astrocytes; Cell culture; Neural stem cell; Neural induction; Neural differentiation; Oligodendrocytes; Neuron; Nestin
16.  Contribution of hepatic lineage stage-specific donor memory to the differential potential of induced mouse pluripotent stem cells (iPSC) 
Stem cells (Dayton, Ohio)  2012;30(5):997-1007.
Recent studies suggested that induced pluripotent stem cells (iPSCs) retain a residual donor cell gene expression which may impact their capacity to differentiate into cell of origin. Here we addressed a contribution of a lineage stage-specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast-derived, HB-iPSCs) and end stage (adult hepatocyte, AH-iPSCs) of hepatocyte differentiation as well as from mouse fetal fibroblasts (MEF-iPSCs) using a lentiviral vector encoding four pluripotency-inducing factors Oct4, Sox2, Klf4, and c-Myc. All resulting iPS cell lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay and normal diploid karyotype. However, HB-iPSCs were more efficient in directed differentiation towards hepatocytic lineage as compared to AH-iPSCs, MEF-iPSCs or mESCs. Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB-iPSCs retained a transcriptional memory (7 up- and 17 down-regulated genes) typical of the original cells. Continuous passaging of HB-iPSCs erased most of these differences including a superior capacity for hepatic re-differentiation. These results suggest that retention of lineage stage-specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set help to improve hepatic differentiation for therapeutic applications and contribute to the better understanding of liver development.
doi:10.1002/stem.1074
PMCID: PMC4017241  PMID: 22378611
induced pluripotent stem cells; donor memory; hepatocyte lineage cells; hepatic differentiation
17.  Astrocytes generated from patient induced pluripotent stem cells recapitulate features of Huntington’s disease patient cells 
Molecular Brain  2012;5:17.
Background
Huntington’s Disease (HD) is a devastating neurodegenerative disorder that clinically manifests as motor dysfunction, cognitive impairment and psychiatric symptoms. There is currently no cure for this progressive and fatal disorder. The causative mutation of this hereditary disease is a trinucleotide repeat expansion (CAG) in the Huntingtin gene that results in an expanded polyglutamine tract. Multiple mechanisms have been proposed to explain the preferential striatal and cortical degeneration that occurs with HD, including non-cell-autonomous contribution from astrocytes. Although numerous cell culture and animal models exist, there is a great need for experimental systems that can more accurately replicate the human disease. Human induced pluripotent stem cells (iPSCs) are a remarkable new tool to study neurological disorders because this cell type can be derived from patients as a renewable, genetically tractable source for unlimited cells that are difficult to acquire, such as neurons and astrocytes. The development of experimental systems based on iPSC technology could aid in the identification of molecular lesions and therapeutic treatments.
Results
We derived iPSCs from a father with adult onset HD and 50 CAG repeats (F-HD-iPSC) and his daughter with juvenile HD and 109 CAG repeats (D-HD-iPSC). These disease-specific iPSC lines were characterized by standard assays to assess the quality of iPSC lines and to demonstrate their pluripotency. HD-iPSCs were capable of producing phenotypically normal, functional neurons in vitro and were able to survive and differentiate into neurons in the adult mouse brain in vivo after transplantation. Surprisingly, when HD-iPSCs were directed to differentiate into an astrocytic lineage, we observed the presence of cytoplasmic, electron clear vacuoles in astrocytes from both F-HD-iPSCs and D-HD-iPSCs, which were significantly more pronounced in D-HD-astrocytes. Remarkably, the vacuolation in diseased astrocytes was observed under basal culture conditions without additional stressors and increased over time. Importantly, similar vacuolation phenotype has also been observed in peripheral blood lymphocytes from individuals with HD. Together, these data suggest that vacuolation may be a phenotype associated with HD.
Conclusions
We have generated a unique in vitro system to study HD pathogenesis using patient-specific iPSCs. The astrocytes derived from patient-specific iPSCs exhibit a vacuolation phenotype, a phenomenon previously documented in primary lymphocytes from HD patients. Our studies pave the way for future mechanistic investigations using human iPSCs to model HD and for high-throughput therapeutic screens.
doi:10.1186/1756-6606-5-17
PMCID: PMC3506453  PMID: 22613578
Huntington’s disease; Induced pluripotent stem cells; Neural differentiation; Astrocytes; Disease modeling
18.  Progress in the reprogramming of somatic cells 
Circulation research  2013;112(3):562-574.
Pluripotent stem cells can differentiate into nearly all types of cells in the body. This unique potential provides significant promise for cell-based therapies to restore tissues or organs destroyed by injuries, degenerative diseases, aging, or cancer. The discovery of induced pluripotent stem cell (iPSC) technology offers a possible strategy to generate patient-specific pluripotent stem cells. However, because of concerns about the specificity, efficiency, kinetics, and safety of iPSC reprogramming, improvements or fundamental changes in this process are required before their effective clinical use. A chemical approach is regarded as a promising strategy to improve and change the iPSC process. Dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve iPSC reprogramming. In addition to the prospect of deriving patient-specific tissues and organs from iPSCs, another attractive strategy for regenerative medicine is transdifferentiation—the direct conversion of one somatic cell type to another. Recent studies revealed a new paradigm of transdifferentiation: using transcription factors (TFs) employed in iPSC generation to induce transdifferentiation, or iPSC TF-based transdifferentiation. This transdifferentiation not only reveals and utilizes the developmentally plastic intermediates generated during iPSC reprogramming, but also produces a very wide range of cells, including expandable tissue-specific precursor cells. Here, we review recent progress of small-molecule approaches in the generation of iPSCs. In addition, we summarize the new concept of iPSC TF–based transdifferentiation and discuss its application in generating various lineage-specific cells, especially cardiovascular cells.
doi:10.1161/CIRCRESAHA.111.249235
PMCID: PMC3790469  PMID: 23371904
Reprogramming; iPSC; small molecule; transdifferentiation and cardiovascular cell
19.  Transient p53 Suppression Increases Reprogramming of Human Fibroblasts without Affecting Apoptosis and DNA Damage 
Stem Cell Reports  2014;3(3):404-413.
Summary
The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion, transient p53 suppression increases reprogramming efficiency without affecting genomic stability, rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation.
Graphical Abstract
Highlights
•Transient p53 suppression increases reprogramming efficiency through p21 inhibition•No adverse effect on DNA damage and apoptosis is observed during reprogramming•Stable iPSC lines display normal karyotypes and expression of pluripotency markers•The iPSC lines retain their differentiation potential and form functional neurons
In this article, Clausen and colleagues show that transient p53 suppression increases reprogramming efficiency in defined conditions without affecting apoptosis and DNA damage. Moreover, iPSC lines generated with or without transient p53 suppression are identical with respect to their pluripotent phenotype and their mutational load and can give rise to functional neurons in vitro.
doi:10.1016/j.stemcr.2014.07.006
PMCID: PMC4266010  PMID: 25241739
20.  Generation of Human Induced Pluripotent Stem Cells Bearing an Anti-HIV Transgene by a Lentiviral Vector Carrying an Internal Murine Leukemia Virus Promoter 
Human Gene Therapy  2010;21(11):1555-1567.
Previously, the Chen laboratory described the generation of a chimeric vector containing a murine leukemia virus (MLV) promoter internal to a lentiviral vector back bone. In this report, the authors report that this chimeric MLV/lentiviral vector can be used to generate induced pluripotent stem (iPS) cells. Kamata et al. demonstrate that these iPS cells are virtually indistinguishable from human embryonic stem cells and are capable of differentiating into several lineages.
Abstract
The recent development of induced pluripotent stem cells (iPSCs) by ectopic expression of defined reprogramming factors offers enormous therapeutic opportunity. To deliver these factors, murine leukemia virus (MLV)-based vectors have been broadly used in the setting of hematopoietic stem cell transplantation. However, MLV vectors have been implicated in malignancy induced by insertional mutagenesis, whereas lentiviral vectors have not. Furthermore, the infectivity of MLV vectors is limited to dividing cells, whereas lentiviral vectors can also transduce nondividing cells. One important characteristic of MLV vectors is a self-silencing property of the promoter element in pluripotent stem cells, allowing temporal transgene expression in a nonpluripotent state before iPSC derivation. Here we test iPSC generation using a novel chimeric vector carrying a mutant MLV promoter internal to a lentiviral vector backbone, thereby containing the useful properties of both types of vectors. Transgene expression of this chimeric vector was highly efficient compared with that of MLV vectors and was silenced specifically in human embryonic stem cells. Human fetal fibroblasts transduced with the vector encoding each factor were efficiently reprogrammed into a pluripotent state, and these iPSCs had potential to differentiate into a variety of cell types. To explore the possibility of iPSCs for gene therapy, we established iPSC clones expressing a short hairpin RNA (shRNA) targeting chemokine receptor 5 (CCR5), the main coreceptor for HIV-1. Using a reporter construct for CCR5 expression, we confirmed that CCR5 shRNA was expressed and specifically knocked down the reporter expression in iPSCs. These data indicate that our chimeric lentiviral vector is a valuable tool for generation of iPSCs and the combination with vectors encoding transgenes allows for rapid establishment of desired genetically engineered iPSC lines.
doi:10.1089/hum.2010.050
PMCID: PMC2978547  PMID: 20524893
21.  Human induced pluripotent stem cell-derived neural stem cells survive, migrate, differentiate, and improve neurologic function in a rat model of middle cerebral artery occlusion 
Introduction
Stroke is a major cause of permanent neurologic damage, with few effective treatments available to restore lost function. Induced pluripotent stem cells (iPSCs) have the potential to generate all cell types in vitro and can be generated from a stroke patient. Therefore, iPSCs are attractive donor sources of genetically identical “patient-specific” cells to hold promise in therapy for stroke. In the present study, we established a four-stage culture system by using serum-free medium and retinoic acid (RA) to differentiate iPSCs into neural stem cells (NSCs) effectively and stably. Our hypothesis was that iPSC-derived NSCs would survive, migrate, and differentiate in vivo, and improve neurologic function after transplantation into the brains of rats with ischemic stroke.
Methods
Human iPSCs (iPS-S-01) and human ESCs (HuES17) were used to differentiate into NSCs by using our four-stage culture system. iPSCs and differentiated NSCs were characterized by immunocytochemistry staining and reverse transcription-polymerase chain reaction (RT-PCR) analysis. After establishment of focal cerebral ischemia with occlusion of the middle cerebral artery (MCA) and cell transplantation, animals were killed at 1 week and 2 weeks to analyze survival, migration, and differentiation of implanted cells in brain tissue. Animal behavior was evaluated via rope grabbing, beam walking, and Morris water maze tests.
Results
iPSCs were efficiently induced into NSCs by using a newly established four-stage induction system in vitro. iPSCs expressed pluripotency-associated genes Oct4, Sox2, and Nanog before NSC differentiation. The iPSC-derived NSCs spontaneously differentiated into neurons and astrocytes, which highly express β-tubulin and glial fibrillary acidic protein (GFAP), respectively. On transplantation into the striatum, CM-DiI labeled iPSC-derived NSCs were found to migrate into the ischemia area at 1 week and 2 weeks, and animal-function recovery was significantly improved in comparison with control groups at 3 weeks.
Conclusions
The four-stage induction system is stable and effective to culture, differentiate, and induce iPSCs to NSCs by using serum-free medium combined with retinoic acid (RA). Implanted iPSC-derived NSCs were able to survive, migrate into the ischemic brain area to differentiate into mature neural cells, and seem to have potential to restore lost neurologic function from damage due to stroke in a rat model.
doi:10.1186/scrt224
PMCID: PMC3706848  PMID: 23769173
Induced pluripotent stem cell; Stoke; Neural stem cell; Middle cerebral artery occlusion
22.  Blood Cell-Derived Induced Pluripotent Stem Cells Free of Reprogramming Factors Generated by Sendai Viral Vectors 
This study demonstrated a reproducible protocol for reprogramming blood cells into transgene-free induced pluripotent stem cells (iPSCs) using Sendai viral vectors. Creation of iPSCs, without integration of exogenous DNA, helps preserve genomic integrity and fosters the development of therapeutic-grade stem cells for regenerative medicine.
The discovery of induced pluripotent stem cells (iPSCs) holds great promise for regenerative medicine since it is possible to produce patient-specific pluripotent stem cells from affected individuals for potential autologous treatment. Using nonintegrating cytoplasmic Sendai viral vectors, we generated iPSCs efficiently from adult mobilized CD34+ and peripheral blood mononuclear cells. After 5–8 passages, the Sendai viral genome could not be detected by real-time quantitative reverse transcription-polymerase chain reaction. Using the spin embryoid body method, we showed that these blood cell-derived iPSCs could efficiently be differentiated into hematopoietic stem and progenitor cells without the need of coculture with either mouse or human stromal cells. We obtained up to 40% CD34+ of which ∼25% were CD34+/CD43+ hematopoietic precursors that could readily be differentiated into mature blood cells. Our study demonstrated a reproducible protocol for reprogramming blood cells into transgene-free iPSCs by the Sendai viral vector method. Maintenance of the genomic integrity of iPSCs without integration of exogenous DNA should allow the development of therapeutic-grade stem cells for regenerative medicine.
doi:10.5966/sctm.2013-0006
PMCID: PMC3726135  PMID: 23847002
CD34+; Reprogramming; Hematopoietic progenitors; Differentiation
23.  Generation of Neural Cells from DM1 Induced Pluripotent Stem Cells As Cellular Model for the Study of Central Nervous System Neuropathogenesis 
Cellular Reprogramming  2013;15(2):166-177.
Abstract
Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study, we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4, hSox2, hKlf4, and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci, a hallmark of DM1, were detected in DM1 iPSCs, neural stem cells (NSCs), and terminally differentiated neurons and astrocytes. In conclusion, we have successfully established disease-specific human DM1 iPSC lines, NSCs, and neuronal lineages with pathognomonic intranuclear RNA foci, which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development.
doi:10.1089/cell.2012.0086
PMCID: PMC3616452  PMID: 23550732
24.  Maintenance and Neuronal Differentiation of Chicken Induced Pluripotent Stem-Like Cells 
Stem Cells International  2014;2014:182737.
Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons.
doi:10.1155/2014/182737
PMCID: PMC4276120  PMID: 25610469
25.  Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells 
Nature protocols  2013;8(9):1670-1679.
Forebrain γ-aminobutyric acid (GABA) interneurons have crucial roles in high-order brain function via modulating network activities and plasticity, and they are implicated in many psychiatric disorders. Availability of enriched functional human forebrain GABA interneurons, especially those from people affected by GABA interneuron deficit disease, will be instrumental to the investigation of disease pathogenesis and development of therapeutics. We describe a protocol for directed differentiation of forebrain GABA interneurons from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a chemically defined system. In this protocol, human PSCs are first induced to primitive neuroepithelial cells over 10 d, and then patterned to NKX2.1-expressing medial ganglionic eminence progenitors by simple treatment with sonic hedgehog or its agonist purmorphamine over the next 2 weeks. These progenitors generate a nearly pure population of forebrain GABA interneurons by the sixth week. This simple and efficient protocol does not require transgenic modification or cell sorting, and it has been replicated with multiple human ESC and iPSC lines.
doi:10.1038/nprot.2013.106
PMCID: PMC4121169  PMID: 23928500

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