• Hippo signaling
• LATS inhibition
• alveolar epithelial type I cells
• directed differentiation
• lung
• lung epithelial reporter
• pluripotent stem cells

• Humans
• Cell Differentiation
• Alveolar Epithelial Cells/cytology
• Alveolar Epithelial Cells/metabolism
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Signal Transduction
• Cells, Cultured
• Transcriptome/genetics
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/metabolism

• U01 TR001810 NCATS NIH HHS
• U01 HL152976 NHLBI NIH HHS
• K08 HL163494 NHLBI NIH HHS
• U01 HL134745 NHLBI NIH HHS
• R01 HL095993 NHLBI NIH HHS
• F31 HL158193 NHLBI NIH HHS
• U01 HL134766 NHLBI NIH HHS
• R01 HL124392 NHLBI NIH HHS
• P01 HL170952 NHLBI NIH HHS

• Dental pulp
• Induced pluripotent stem cells
• Klf4
• Oct3/4
• Sendai virus
• Sox2
• c-Myc

• Humans
• Induced Pluripotent Stem Cells
• Sendai virus/genetics
• Cell Differentiation/genetics
• Cell Lineage
• Drug Discovery

• U01 TR001810 NCATS NIH HHS
• U01 HL152976 NHLBI NIH HHS
• T32 HL007035 NHLBI NIH HHS
• U01 HL134745 NHLBI NIH HHS
• R01 HL095993 NHLBI NIH HHS
• F31 HL158193 NHLBI NIH HHS
• R01 HL124392 NHLBI NIH HHS
• UL1 TR001430 NCATS NIH HHS
• U01 HL134766 NHLBI NIH HHS
• 75N92020C00005 NHLBI NIH HHS

The induced pluripotent stem cells (iPSCs) are considered powerful tools in pharmacology, biomedicine, toxicology, and cell therapy. Multiple approaches have been used to generate iPSCs with the expression of reprogramming factors. Here, we generated iPSCs by integrating the reprogramming cassette into a genomic safe harbor, CASH-1, with the use of a precise genome editing tool, CRISPR/Cas9. The integration of cassette at CASH-1 into target cells did not alter the pattern of proliferation and interleukin-6 secretion as a response to ligands of multiple signaling pathways involving tumor necrosis factor-α receptor, interleukin-1 receptor, and toll-like receptors. Moreover, doxycycline-inducible expression of OCT4, SOX2, and KLF4 reprogrammed engineered human dermal fibroblasts and human embryonic kidney cell line into iPSCs. The generated iPSCs showed their potential to make embryoid bodies and differentiate into the derivatives of all three germ layers. Collectively, our data emphasize the exploitation of CASH-1 by CRISPR/Cas9 tool for therapeutic and biotechnological applications including but not limited to reprogramming of engineered cells into iPSCs.

To master the technology of reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs), which will lay a good foundation for setting up a technology platform on reprogramming human cancer cells into iPSCs. Mouse iPSCs (i.e., Oct4-GFP miPSCs) was successfully generated from mouse embryonic fibroblasts (MEFs) harboring Oct4-EGFP transgene by introducing four factors, Oct4, Sox2, c-Myc and Klf4, under mESC (Murine embryonic stem cells) culture conditions. Oct4-GFP miPSCs were similar to mESCs in morphology, proliferation, mESC-specific surface antigens and gene expression. Additionally, Oct4-GFP miPSCs could be cultured in suspension to form embryoid bodies (EBs) and differentiate into cell types of the three germ layers in vitro. Moreover, Oct4-GFP miPSCs could develop to teratoma and chimera in vivo. Unlike cell cycle distribution of MEFs, Oct4-GFP miPSCs are similar to mESCs in the cell cycle structure which consists of higher S phase and lower G1 phase. More importantly, our data demonstrated that MEFs harboring Oct4-EGFP transgene did not express GFP, until they were reprogrammed to the pluripotent stage (iPSCs), while the GFP expression was progressively lost when these pluripotent Oct4-GFP miPSCs exposed to EB-mediated differentiation conditions, suggesting the pluripotency of Oct4-GFP miPSCs can be real-time monitored over long periods of time via GFP assay. Altogether, our findings demonstrate that Oct4-GFP miPSC line is successfully established, which will lay a solid foundation for setting up a technology platform on reprogramming cancer cells into iPSCs. Furthermore, this pluripotency reporter system permits the long-term real-time monitoring of pluripotency changes in a live single-cell, and its progeny.

• Oct4-EGFP transgene
• cancer cell reprogramming
• cancer therapy
• induced pluripotent stem (iPS) cells
• pluripotency reporter system

• 3D cell culture systems
• Cardiac regeneration
• Cardiovascular disease modeling
• Induced pluripotent stem cells
• Precision medicine

• Cardiovascular Diseases/metabolism
• Cardiovascular Diseases/therapy
• Cell Differentiation/physiology
• Endothelial Cells
• Humans
• Induced Pluripotent Stem Cells
• Myocytes, Cardiac

• R01 HL141851 NIH HHS
• R01 HL150693 NIH HHS
• R01 HL145676 NIH HHS
• R01 HL123968 NHLBI NIH HHS
• R01 HL123968 NIH HHS
• R01 HL150693 NHLBI NIH HHS
• R01 HL145676 NHLBI NIH HHS
• R01 HL141851 NHLBI NIH HHS

• Cre-loxP system
• hepatic organoids
• hepatocyte differentiation
• hiPSCs
• transgene-free

• 50535-2021 National Research Foundation of Korea
• 50674-2021 of NRF-2019R1F1A1060636 National Research Foundation of Korea
• 2020R1I1A1A01070097 National Research Foundation of Korea
• 21A0401L1-11 of 2021M3E5E5095559 Korean Fund for Regenerative Medicine, funded by the Ministry of Science and ICT, and the Ministry of Health and Welfare

Self-limited Childhood Epilepsies are the most prevalent epileptic syndrome in children. Its pathogenesis is unknown. In this disease, symptoms resolve spontaneously in approximately 50% of patients when maturity is reached, prompting to a maturation problem. The purpose of this study was to understand the molecular bases of this disease by generating and analyzing induced pluripotent stem cell-derived neurons from a family with 7 siblings, among whom 4 suffer from this disease.

Two affected siblings and, as controls, a healthy sister and the unaffected mother of the family were studied. Using exome sequencing, a homozygous variant in the FYVE, RhoGEF and PH Domain Containing 6 gene was identified in the patients as a putative genetic factor that could contribute to the development of this familial disorder. After informed consent was signed, skin biopsies from the 4 individuals were collected, fibroblasts were derived and reprogrammed and neurons were generated and characterized by markers and electrophysiology. Morphological, electrophysiological and gene expression analyses were performed on these neurons.

Bona fide induced pluripotent stem cells and derived neurons could be generated in all cases. Overall, there were no major shifts in neuronal marker expression among patient and control-derived neurons. Compared to two familial controls, neurons from patients showed shorter axonal length, a dramatic reduction in synapsin-1 levels and cytoskeleton disorganization. In addition, neurons from patients developed a lower action potential threshold with time of in vitro differentiation and the amount of current needed to elicit an action potential (rheobase) was smaller in cells recorded from NE derived from patients at 12 weeks of differentiation when compared with shorter times in culture. These results indicate an increased excitability in patient cells that emerges with the time in culture. Finally, functional genomic analysis showed a biased towards immaturity in patient-derived neurons.

We are reporting the first in vitro model of self-limited childhood epilepsy, providing the cellular bases for future in-depth studies to understand its pathogenesis. Our results show patient-specific neuronal features reflecting immaturity, in resonance with the course of the disease and previous imaging studies.

The online version contains supplementary material available at 10.1186/s13287-021-02658-2.

Cell-based therapy (CBT) is attracting much attention to treat incurable diseases. In recent years, several clinical trials have been conducted using human pluripotent stem cells (hPSCs), and other potential therapeutic cells. Various private- and government-funded organizations are investing in finding permanent cures for diseases that are difficult or expensive to treat over a lifespan, such as age-related macular degeneration, Parkinson’s disease, or diabetes, etc. Clinical-grade cell manufacturing requiring current good manufacturing practices (cGMP) has therefore become an important issue to make safe and effective CBT products. Current cell production practices are adopted from conventional antibody or protein production in the pharmaceutical industry, wherein cells are used as a vector to produce the desired products. With CBT, however, the “cells are the final products” and sensitive to physico- chemical parameters and storage conditions anywhere between isolation and patient administration. In addition, the manufacturing of cellular products involves multi-stage processing, including cell isolation, genetic modification, PSC derivation, expansion, differentiation, purification, characterization, cryopreservation, etc. Posing a high risk of product contamination, these can be time- and cost- prohibitive due to maintenance of cGMP. The growing demand of CBT needs integrated manufacturing systems that can provide a more simple and cost-effective platform. Here, we discuss the current methods and limitations of CBT, based upon experience with biologics production. We review current cell manufacturing integration, automation and provide an overview of some important considerations and best cGMP practices. Finally, we propose how multi-stage cell processing can be integrated into a single bioreactor, in order to develop streamlined cGMP-compliant cell processing systems.

• biologics manufacturing
• bioreactor
• cGMP
• cell-based therapy
• genetic engineering
• integrated bioprocessing

• Episomal reprogramming
• Human peripheral mononuclear cells
• Induced pluripotent stem cells
• Stem cells
• iPSC

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) has opened new therapeutic possibilities. However, karyotypic abnormalities detected in iPSCs compromised their utility, especially chromosomal aberrations found at early passages raised serious safety concerns. The mechanism underlying the chromosomal abnormality in early-passage iPSCs is not known.

Human dermal fibroblasts (HDFs) were stimulated with KMOS (KLF4, cMYC, OCT4 and SOX2) proteins to enhance their proliferative capacity and many vigorous clones were obtained. Clonal reprogramming was carried out by KMOS mRNAs transfection to confirm the ‘chromosomal mutagenicity’ of reprogramming process. Subculturing was performed to examine karyotypic stability of iPSCs after the re-establishment of stemness. And antioxidant N-acetyl-cysteine (NAC) was added to the culture medium for further confirmming the mutagenicity in the first few days of reprogramming.

Chromosomal aberrations were found in a small percentage of newly induced iPS clones by reprogramming transcription factors. Clonal reprogramming ruled out the aberrant chromosomes inherited from rare karyotypically abnormal parental cell subpopulation. More importantly, the antioxidant NAC effectively reduced the occurrence of chromosomal aberrations at the early stage of reprogramming. Once iPS cell lines were established, they restored karyotypic stability in subsequent subculturing.

Our results provided the first line of evidence for the ‘chromosomal mutagenicity’ of reprogramming process.

• Chromosomal aberration
• Genetic stability
• Induced pluripotent stem cell
• Karyotype
• Reprogramme

During somatic reprogramming, Yamanaka’s pioneer factors regulate a complex sequence of molecular events leading to the activation of a network of pluripotency factors, ultimately resulting in the acquisition and maintenance of a pluripotent state. Here, we show that, contrary to the pluripotency factors studied so far, overexpression of Mybl2 inhibits somatic reprogramming. Our results demonstrate that Mybl2 levels are crucial to the dynamics of the reprogramming process. Mybl2 overexpression changes chromatin conformation, affecting the accessibility of pioneer factors to the chromatin and promoting accessibility for early immediate response genes known to be reprogramming blockers. These changes in the chromatin landscape ultimately lead to a deregulation of key genes that are important for the mesenchymal-to-epithelial transition. This work defines Mybl2 level as a gatekeeper for the initiation of reprogramming, providing further insights into the tight regulation and required coordination of molecular events that are necessary for changes in cell fate identity during the reprogramming process.

•

Deletion and overexpression of MYBL2 pluripotency factor inhibit somatic reprogramming

• AP1
• ATAC-sequencing
• Jun
• Sox2
• chromatin landscape
• chromatin remodeling
• induced pluripotent stem cells
• mesenchymal-to-epithelial transition
• reprogramming blockers
• somatic reprogramming

• Episomal
• Reprogramming
• Sendai virus
• hiPSCs

• Animals
• Cellular Reprogramming
• Cellular Reprogramming Techniques/methods
• HEK293 Cells
• HL-60 Cells
• HeLa Cells
• Hep G2 Cells
• Humans
• Induced Pluripotent Stem Cells/cytology
• Induced Pluripotent Stem Cells/metabolism
• Plasmids/genetics
• Plasmids/metabolism
• Sendai virus/genetics
• Sendai virus/metabolism

• U54 DK110805 NIDDK NIH HHS

• Collagen
• Differentiation
• Induced pluripotent stem cells
• Integrin
• Reprogramming
• Smooth muscle cells

• STEMCCA lentiviral vector
• Sendai virus vector
• amniocytes
• feeder-free reprogramming system
• human induced pluripotent stem cells (hiPSCs)
• peripheral blood mononuclear cells (PBMCs)

• autologous
• induced pluripotent stem cells
• retinal degeneration
• stem cell

Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

• OCT4
• chitosan nanoparticles
• induced pluripotent stem cells
• reprogramming
• transgene-free stem cells

• FTD/ALS
• amyotrophic lateral sclerosis
• dementia modeling
• frontotemporal dementia
• induced pluripotent stem cells

Since the embryonic stem cells have knocked the doorsteps, they have proved themselves in the field of science, research, and medicines, but the hovered restrictions confine their application in human welfare. Alternate approaches used to reprogram the cells to the pluripotent state were not up to par, but the innovation of induced pluripotent stem cells (iPSCs) paved a new hope for the researchers. Soon after the discovery, iPSCs technology is undergoing renaissance day by day, i.e., from the use of genetic material to recombinant proteins and now only chemicals are employed to convert somatic cells to iPSCs. Thus, this technique is moving straightforward and productive at an astonishing pace. Here, we provide a brief introduction to iPSCs, the mechanism and methods for their generation, their prevailing and prospective applications and the future opportunities that can be expected from them.

• cellular reprogramming
• embryonic stem cells
• induced pluripotent stem cells
• stem cells

• T cell
• biomanufacturing
• cell therapy
• scale-out
• scale-up
• stem cell

• induced pluripotent stem cells (iPSCs)
• non-human primate
• reprogramming
• rhesus macaque

• Marfan syndrome
• disease modeling
• genome editing
• human pluripotent stem cells
• osteogenesis
• smooth muscle.

• Animals
• Bone and Bones/cytology
• Cell Differentiation/physiology
• Humans
• Induced Pluripotent Stem Cells/cytology
• Mesenchymal Stem Cells/cytology
• Osteogenesis/physiology
• Tissue Engineering/methods

The cellular conditions required to establish induced pluripotent stem cells (iPSCs), such as the number of reprogramming factors and/or promoter selection, differ among species. The establishment of iPSCs derived from cells of previously unstudied species therefore requires the extensive optimization of programming conditions, including promoter selection and the optimal number of reprogramming factors, through a trial-and-error approach. While the four Yamanaka factors Oct3/4, Sox2, Klf4, and c-Myc are sufficient for iPSC establishment in mice, we reported previously that six reprogramming factors were necessary for the creation of iPSCs from primary prairie vole-derived cells. Further to this study, we now show detailed data describing the optimization protocol we developed in order to obtain iPSCs from immortalized prairie vole-derived fibroblasts. Immortalized cells can be very useful tools in the optimization of cellular reprogramming conditions, as cellular senescence is known to dramatically decrease the efficiency of iPSC establishment. The immortalized prairie vole cells used in this optimization were designated K4DT cells as they contained mutant forms of CDK4, cyclin D, and telomerase reverse transcriptase (TERT). We show that iPSCs derived from these immortalized cells exhibit the transcriptional silencing of exogenous reprogramming factors while maintaining pluripotent cell morphology. There were no observed differences between the iPSCs derived from primary and immortalized prairie vole fibroblasts. Our data suggest that cells that are immortalized with mutant CDK4, cyclin D, and TERT provide a useful tool for the determination of the optimal conditions for iPSC establishment.

• Cellular senescence
• Immortalized cells
• Induced pluripotent stem cell (iPSC)
• Pluripotency
• Prairie vole

• differentiation
• hepatocytes
• pluripotent cells

Low efficiency of somatic cell reprogramming and heterogeneity among human induced pluripotent stem cells (hiPSCs) demand extensive characterization of isolated clones before their use in downstream applications. By monitoring human fibroblasts undergoing reprogramming for their morphological changes and expression of fibroblast (CD13), pluripotency markers (SSEA-4 and TRA-1-60) and a retrovirally expressed red fluorescent protein (RV-RFP), we compared the efficiency of these features to identify bona fide hiPSC colonies. The co-expression kinetics of fibroblast and pluripotency markers in the cells being reprogrammed and the emerging colonies revealed the heterogeneity within SSEA-4⁺ and TRA-1-60⁺ cells, and the inadequacy of these commonly used pluripotency markers for the identification of bona fide hiPSC colonies. The characteristic morphological changes in the emerging hiPSC colonies derived from fibroblasts expressing RV-RFP showed a good correlation between hiPSC morphology acquisition and silencing of RV-RFP and facilitated the easy identification of hiPSCs. The kinetics of retroviral silencing and pluripotency marker expression in emerging colonies suggested that combining both these markers could demarcate the stages of reprogramming with better precision than with pluripotency markers alone. Our results clearly demonstrate that the pluripotency markers that are routinely analyzed for the characterization of established iPSC colonies are not suitable for the isolation of pluripotent cells in the early stages of reprogramming, and silencing of retrovirally expressed reporter genes helps in the identification of colonies that have attained a pluripotent state and the morphology of human embryonic stem cells (hESCs).

Summary: The use of hESC-like morphology, retroviral transgene silencing and temporal expression of pluripotency markers are compared as methods to aid in the identification of hiPSC clones.

• Colony identification
• Human induced pluripotent stem cells
• Pluripotency
• Reprogramming
• Retroviral silencing
• hiPSC morphology

• Beta-thalassemia
• Cell therapy
• Gene therapy
• Induced pluripotent stem cells
• Reprogramming
• Sickle cell disease

• Human induced pluripotent stem cells
• Reprogramming

• H3K4 methyltransferase
• MLL4/KMT2D
• cell fate transition
• enhancer
• p300

• Differentiation
• Foetal cells
• Gene editing
• Gene therapy
• Human induced pluripotent stem cells
• Monogenic diseases
• Reprogramming techniques

Induced pluripotent stem cells (iPSC) are a most promising approach to the development of a hepatocyte transplantable mass sufficient to induce long-term correction of inherited liver metabolic diseases, thus avoiding liver transplantation. Their intrinsic self-renewal ability and potential to differentiate into any of the three germ layers identify iPSC as the most promising cell-based therapeutics, but also as drivers of tumor development. Teratoma development currently represents the gold standard to assess iPSC pluripotency. We analyzed the tumorigenic potential of iPSC generated from human hepatocytes (HEP-iPSC) and compared their immunohistochemical profiles to that of tumors developed from fibroblast and hematopoietic stem cell-derived iPSC. HEP-iPSC generated tumors significantly presented more malignant morphological features than reprogrammed fibroblasts or CD34+ iPSC. Moreover, the protooncogene myc showed the strongest expression in HEP-iPSC, compared to only faint expression in the other cell subsets. Random integration of transgenes and the use of potent protooncogenes such as myc might be a risk factor for malignant tumor development if hepatocytes are used for reprogramming. Nonviral vector delivery systems or reprogramming of cells obtained from less invasive harvesting methods would represent interesting options for future developments in stem cell-based approaches for liver metabolic diseases.

An attempt was made to reprogram peripheral blood cells into human induced pluripotent stem cell (hiPSCs) as a new cell source for cartilage repair.

We generated chondrogenic lineage from human peripheral blood via hiPSCs using an integration-free method. Peripheral blood cells were either obtained from a human blood bank or freshly collected from volunteers. After transforming peripheral blood cells into iPSCs, the newly derived iPSCs were further characterized through karyotype analysis, pluripotency gene expression and cell differentiation ability. iPSCs were differentiated through multiple steps, including embryoid body formation, hiPSC-mesenchymal stem cell (MSC)-like cell expansion, and chondrogenic induction for 21 days. Chondrocyte phenotype was then assessed by morphological, histological and biochemical analysis, as well as the chondrogenic expression.

hiPSCs derived from peripheral blood cells were successfully generated, and were characterized by fluorescent immunostaining of pluripotent markers and teratoma formation in vivo. Flow cytometric analysis showed that MSC markers CD73 and CD105 were present in monolayer cultured hiPSC–MSC-like cells. Both alcian blue and toluidine blue staining of hiPSC–MSC-chondrogenic pellets showed as positive. Immunohistochemistry of collagen II and X staining of the pellets were also positive. The sulfated glycosaminoglycan content was significantly increased, and the expression levels of the chondrogenic markers COL2, COL10, COL9 and AGGRECAN were significantly higher in chondrogenic pellets than in undifferentiated cells. These results indicated that peripheral blood cells could be a potential source for differentiation into chondrogenic lineage in vitro via generation of mesenchymal progenitor cells.

This study supports the potential applications of utilizing peripheral blood cells in generating seed cells for cartilage regenerative medicine in a patient-specific and cost-effective approach.

The online version of this article (doi:10.1186/s13287-016-0290-7) contains supplementary material, which is available to authorized users.

• cystic fibrosis ips cells
• ipsc directed differentiation
• polynucleotide gene targeting/genome editing
• sequence-specific chimeric endonucleases
• sfhr

• T32 DK007636 NIDDK NIH HHS
• P30 DK072517 NIDDK NIH HHS
• R01 DK104681 NIDDK NIH HHS
• PN2 EY018244 NEI NIH HHS
• P01 DK088760 NIDDK NIH HHS
• R01 GM075111 NIGMS NIH HHS

• Cardiac differentiation
• Induced pluripotent stem cells
• Transcriptional profile

• Animals
• Cadherins/genetics
• Cadherins/metabolism
• Cell Differentiation
• Cells, Cultured
• Cytochrome P-450 Enzyme System/genetics
• Cytochrome P-450 Enzyme System/metabolism
• Embryo, Mammalian/cytology
• Fibroblasts/cytology
• Fibroblasts/metabolism
• GATA4 Transcription Factor/genetics
• Hepatocyte Nuclear Factor 1-alpha/genetics
• Hepatocyte Nuclear Factor 3-gamma/genetics
• Hepatocytes/cytology
• Hepatocytes/metabolism
• Hydrolases/deficiency
• Hydrolases/genetics
• Karyotyping
• Keratin-18/genetics
• Keratin-18/metabolism
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Microscopy, Fluorescence
• Serum Albumin/genetics
• Serum Albumin/metabolism
• Zonula Occludens-1 Protein/genetics
• Zonula Occludens-1 Protein/metabolism

• Human induced pluripotent stem cells
• Reprogramming

To improve the recovery of damaged cartilage tissue, pluripotent stem cell-based therapies are being intensively explored. A number of techniques exist that enable monitoring of stem cell differentiation, including immunofluorescence staining. This simple and fast method enables changes to be observed during the differentiation process. Here, two protocols for the differentiation of human embryonic stem cells into chondrocytes were used (monolayer cell culture and embryoid body formation). Cells were labeled for markers expressed during the differentiation process at different time points (pluripotent: NANOG, SOX2, OCT3/4, E-cadherin; prochondrogenic: SOX6, SOX9, Collagen type II; extracellular matrix components: chondroitin sulfate, heparan sulfate; beta-catenin, CXCR4, and Brachyury). Comparison of the signal intensity of differentiated cells to control cell populations (articular cartilage chondrocytes and human embryonic stem cells) showed decreased signal intensities of pluripotent markers, E-cadherin and beta-catenin. Increased signal intensities of prochondrogenic markers and extracellular matrix components were observed. The changes during chondrogenic differentiation monitored by evaluation of pluripotent and chondrogenic markers signal intensity were described. The changes were similar to several studies over chondrogenesis. These results were confirmed by semi-quantitative analysis of IF signals. In this research we indicate a bioimaging as a useful tool to monitor and semi-quantify the IF pictures during the differentiation of hES into chondrocyte-like.

• Cartilage injuries
• Chondrogenesis
• Immunofluorescence
• Osteoarthritis
• Regenerative medicine

• Induced pluripotent stem cell
• Neurological diseases
• Neuron
• Parkinson’s disease

• expression vectors
• neuroanatomy
• neurotropic virus
• review
• transsynaptic tracing

• Dental pulp stem cells
• Exfoliated teeth
• Spontaneously immortalized

• Animals
• Cell Differentiation/genetics
• Cell Transformation, Neoplastic
• Dental Pulp/cytology
• Dental Pulp/transplantation
• Humans
• Mice
• Neural Crest/transplantation
• Neurons/cytology
• Stem Cell Transplantation/adverse effects
• Telomerase/pharmacology

• R21 NS075709 NINDS NIH HHS
• R21NS075709-02 NINDS NIH HHS

Since the discovery of induced pluripotent stem cells (iPSC) in 2006, the symptoms of many human diseases have been reversed in animal models with iPSC therapy, setting the stage for future clinical development. From the animal data it is clear that iPSC are rapidly becoming the lead cell type for cell replacement therapy and for the newly developing field of iPSC-derived body organ transplantation. The first human pathology that might be treated in the near future with iPSC is age-related macular degeneration (AMD), which has recently passed the criteria set down by regulators for phase I clinical trials with allogeneic human embryonic stem cell-derived cell transplantation in humans. Given that iPSC are currently in clinical trial in Japan (RIKEN) to treat AMD, the establishment of a set of international criteria to make clinical-grade iPSC and their differentiated progeny is the next step in order to prepare for future autologous cell therapy clinical trials. Armed with clinical-grade iPSC, we can then specifically test for their threat of cancer, for proper and efficient differentiation to the correct cell type to treat human disease and then to determine their immunogenicity. Such a rigorous approach sets a far more relevant paradigm for their intended future use than non-clinical-grade iPSC. This review focuses on the latest developments regarding the first possible use of iPSC-derived retinal pigment epithelial cells in treating human disease, covers data gathered on animal models to date and methods to make clinical-grade iPSC, suggests techniques to ensure quality control and discusses possible clinical immune responses.

Down syndrome (DS, trisomy 21), is the most common viable chromosomal disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. Several models have been used to investigate the mechanisms by which the extra copy of chromosome 21 leads to the DS phenotype. In the last five years, several laboratories have been successful in reprogramming patient cells carrying the trisomy 21 anomaly into induced pluripotent stem cells, i.e., T21-iPSCs. In this review, we summarize the different T21-iPSCs that have been generated with a particular interest in the technical procedures and the somatic cell types used for the reprogramming.

• Down syndrome
• T21-iPSCs
• induced pluripotent stem cells
• isogenic iPSCs
• reprogramming
• trisomy 21

Neural stem cell (NSC) transplantation is a promising strategy for delivering therapeutic proteins in the brain. We evaluated a complete process of ex vivo gene therapy using human induced pluripotent stem cell (iPSC)-derived NSC transplants in a well-characterized mouse model of a human lysosomal storage disease, Sly disease. Human Sly disease fibroblasts were reprogrammed into iPSCs, differentiated into a stable and expandable population of NSCs, genetically corrected with a transposon vector, and assessed for engraftment in NOD/SCID mice. Following neonatal intraventricular transplantation, the NSCs engraft along the rostrocaudal axis of the CNS primarily within white matter tracts and survive for at least 4 months. Genetically corrected iPSC-NSCs transplanted post-symptomatically into the striatum of adult Sly disease mice reversed neuropathology in a zone surrounding the grafts, while control mock-corrected grafts did not. The results demonstrate the potential for ex vivo gene therapy in the brain using human NSCs from autologous, non-neural tissues.

•

Sly disease patient fibroblasts converted to iPSCs yield transplantable NSCs