hiPSC Clones Research Articles

Differential X Chromosome Inactivation Patterns during the Propagation of Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) represent a potentially useful tool for studying the molecular mechanisms of disease thanks to their ability to generate patient-specific hiPSC clones. However, previous studies have reported that DNA methylation profiles, including those for imprinted genes, may change during passaging of hiPSCs. This is particularly problematic for hiPSC models of X-linked disease, because unstable X chromosome inactivation status may affect the detection of phenotypes. In the present study, we examined the epigenetic status of hiPSCs derived from patients with Rett syndrome, an X-linked disease, during long-term culture. To analyze X chromosome inactivation, we used a methylation-specific polymerase chain reaction (MSP) to assay the human androgen receptor locus (HUMARA). We found that single cell-derived hiPSC clones exhibit various states of X chromosome inactivation immediately after clonal isolation, even when established simultaneously from a single donor. X chromosome inactivation states remain variable in hiPSC clones at early passages, and this variability may affect cellular phenotypes characteristic of X-linked diseases. Careful evaluation of X chromosome inactivation in hiPSC clones, particularly in early passages, by methods such as HUMARA-MSP, is therefore important when using patient-specific hiPSCs to model X-linked disease.

Human Induced Pluripotent Stem Cells Incorporating Safe Harbor Loci Integrated Inducible Suicide Systems for Use in the Application of Cellular Therapeutics

As the field of human induced pluripotent stem cell (hiPSC) research continues to advance, and as the clinical investigation of genetically-engineered hiPSC-derived cellular therapeutics begins to emerge, safety concerns relating to the administration of genetically-altered cells must be addressed and mitigated. A number of strategies including recombinant peptides, monoclonal antibodies, small molecule-modulated enzyme activity and gene-specific modifications have been explored to facilitate the selective elimination of aberrant cells. Of these, the application of genetically-encoded inducible "suicide" systems that can be rapidly activated by a specific non-toxic chemical inducer represents a very attractive, targeted approach for eliminating administered cells without damaging surrounding cells and tissues. Most previous studies have employed viral vectors and short promoters to stably introduce suicide-genes, including herpes complex virus thymidine kinase or inducible caspase-9 (iCasp9), into human cells. The use of viral vectors can lead to random integration events which can disrupt or activate disease-related genes, potentially causing deleterious effects. In addition, many artificial promoters and genome regions are prone to epigenetic silencing in both pluripotent and differentiated states, resulting in cells becoming unresponsive to suicide gene induction. Thus, it is of great importance to identify optimal integration sites and promoters in order to maintain functional suicide gene responses and facilitate the development of genetically-engineered cellular therapeutics.To effectively select and test suicide systems under the control of various promoters in combination with different safe harbor loci integration strategies, we took advantage of our proprietary hiPSC platform, which enables single cell passaging and high-throughput, 96-well plate-based flow cytometry sorting. In a single hiPSC per well manner, we utilized both nuclease-independent and nuclease-dependent strategies to efficiently and precisely integrate various suicide gene expression cassettes in AAVS1 or ROSA26 safe harbor loci. Several integration vectors, each containing a suicide gene expression cassette downstream of various exogenous and endogenous promoters, including endogenous AAVS1 or ROSA26, cytomegalovirus, elongation factor 1α, phosphoglycerate kinase, hybrid CMV enhancer/chicken β-actin and ubiquitin C promoters, were tested to systematically analyze and compare the activity of different suicide systems in both hiPSCs and hiPSC-derived differentiated cells. To conduct high-throughput analyses of these integration and expression strategies, we selected an iCasp9 suicide gene platform where rapid caspase-9 mediated cell death can be induced by small molecule chemical inducers of dimerization such as AP1903. Several endogenous promoters were found to drive persistent expression of iCasp9 during clonal expansion of hiPSCs, but the expression level was determined to be too low to effectively respond to AP1903 treatment. Expression of iCasp9 under the control of various exogenous promoters was lost during prolonged clonal expansion of hiPSCs, and failed to drive AP1903-induced cell death. However, one promoter maintained high levels of iCasp9 expression during the long-term clonal expansion of hiPSCs. Furthermore, these iCasp9-integrated clonal lines underwent rapid cell death in the presence of AP1903, and no residual cell survival was observed when cultures were allowed to recover in the absence of the dimerizing molecule. To test whether epigenetic landscape alterations would abrogate suicide gene-mediated response, hiPSC clones were differentiated into three somatic lineages in vitro and were found to be completely subject to AP1903-induced cell death. Clones were also specifically differentiated towards hematopoietic cells to demonstrate complete induction of cell death by AP1903 treatment. When injected into NSG mice to form teratomas, similar cell death-mediated response was observed in vivo. Notably, one hiPSC clone contained certain rare cells and did escape induced cell death, and this clone and these rare cells were characterized to assess the molecular mechanisms of escape. Our study describes novel findings toward designing optimal safety systems for integration into hiPSC-derived cellular therapies. DisclosuresHuang:Fate Therapeutics Inc: Employment. Lan:Fate Therapeutics Inc: Employment. Parone:Fate Therapeutics Inc: Employment. Clarke:Fate Therapeutics Inc: Employment. Abujarour:Fate Therapeutics Inc: Employment. Robinson:Fate Therapeutics Inc: Employment. Meza:Fate Therapeutics Inc: Employment. Lee:Fate Therapeutics Inc: Employment. Shoemaker:Fate Therapeutics Inc: Employment. Valamehr:Fate Therapeutics Inc: Employment.

Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.

BackgroundHuman pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves.ResultsIn this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6+ and SOX1+ neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9+ and ISL-1+ motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9e438::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis.ConclusionsOur motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0172-4) contains supplementary material, which is available to authorized users.

Abstract 17739: Modelling Long-QT Syndrome Associated With a Calmodulin Mutation Using Human Induced Pluripotent Stem Cells

Background: Calmodulin ( CALM ) is a ubiquitous Ca2+-sensor molecule, and it modulates various proteins including several ion channels, ryanodine receptor 2, and Ca 2+ /calmodulin-dependent protein kinase. Recently, several studies reported that CALM mutations are associated with severe early-onset long-QT syndrome (LQTS). However, the underlying mechanism remains unknown. Purpose: The present study aims to establish the in-vitro disease model of CALM -related LQTS and elucidate the pathophysiological mechanism using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods: The hiPSC clones were generated from a 12-year-old boy with LQTS carrying a missense CALM2 mutation (c.293A>G, p.N98S), and differentiated into cardiomyocytes via embryoid body (EB) formation. The electrophysiological characteristics of CALM2 -N98S hiPSC-CMs, including action potential (AP) and L-type Ca 2+ channel (LTCC) currents, were analyzed by patch-clamp technique, and compared with those of hiPSC-CMs derived from healthy control. Results: The beating rate of CALM2 -N98S EBs was significantly lower than that of control (23.8 ± 1.6 bpm vs 47.3 ± 1.7 bpm, p < 0.01). The corrected AP duration at 90% repolarization (cAPD90) of CALM2 -N98S hiPSC-CMs was significantly prolonged compared to that of control (480.9 ± 59.4 ms vs 201.5 ± 19.5 ms, p < 0.01, Figure). At 1 Hz pacing, CALM2 -N98S hiPSC-CMs exhibited significantly longer APD90 than control (495.5 ± 47.7 ms vs 227.8 ± 21.6 ms, p < 0.01). The time constants of LTCC inactivation tau fast of CALM2 -N98S hiPSC-CMs were significantly larger than those of control at 0, +10, and +20 mV test potentials (Figure). Conclusion: The hiPSC-CMs model of CALM2 -related LQTS successfully recapitulated the disease phenotypes, and elucidated the pathophysiological mechanism: impaired inactivation of LTCC currents. This model might be useful for drug discovery in CALM2 -related LQTS.

Induction of pluripotency in human umbilical cord mesenchymal stem cells in feeder layer-free condition.

Induced Pluripotent Stem Cells (iPSCs) has been produced by the reprogramming of several types of somatic cells through the expression of different sets of transcription factors. This study consists of a technique to obtain iPSCs from human umbilical cord mesenchymal stem cells (UC-MSCs) in a feeder layer-free process using a mini-circle vector containing defined reprogramming genes, Lin28, Nanog, Oct4 and Sox2. The human MSCs transfected with the minicircle vector were cultured in iPSCs medium. Human embryonic stem cell (ESC)-like colonies with tightly packed domelike structures appeared 7-10 days after the second transfection. In the earliest stages, the colonies were green fluorescence protein (GFP)-positive, while upon continuous culture and passage, genuine hiPSC clones expressing GFP were observed. The induced cells, based on the ectopic expression of the pluripotent markers, exhibited characteristics similar to the embryonic stem cells. These iPSCs demonstrated in vitro capabilities for differentiation into the three main embryonic germ layers by embryoid bodies formation. There was no evidence of transgenes integration into the genome of the iPSCs in this study. In conclusion, this method offers a means of producing iPSCs without viral delivery that could possibly overcome ethical concerns and immune rejection in the use of stem cells in medical applications.

Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells.

CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5–8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

Targeted Gene Correction Minimally Impacts Whole-Genome Mutational Load in Human-Disease-Specific Induced Pluripotent Stem Cell Clones

The utility of genome editing technologies for disease modeling and developing cellular therapies has been extensively documented, but the impact of these technologies on mutational load at the whole-genome level remains unclear. We performed whole-genome sequencing to evaluate the mutational load at single-base resolution in individual gene-corrected human induced pluripotent stem cell (hiPSC) clones in three different disease models. In single-cell clones, gene correction by helper-dependent adenoviral vector (HDAdV) or Transcription Activator-Like Effector Nuclease (TALEN) exhibited few off-target effects and a low level of sequence variation, comparable to that accumulated in routine hiPSC culture. The sequence variants were randomly distributed and unique to individual clones. We also combined both technologies and developed a TALEN-HDAdV hybrid vector, which significantly increased gene-correction efficiency in hiPSCs. Therefore, with careful monitoring via whole-genome sequencing it is possible to apply genome editing to human pluripotent cells with minimal impact on genomic mutational load.

Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.

Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

SummaryGenetic engineering of human induced pluripotent stem cells (hiPSCs) via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs). Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications.

Targeting of Herpes Simplex Virus 1 Thymidine Kinase Gene Sequences into the OCT4 Locus of Human Induced Pluripotent Stem Cells

The in vitro differentiation of human induced pluripotent stem cells (hiPSC) to generate specific types of cells is inefficient, and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC, we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK) suicide gene at the endogenous OCT4 (POU5F1) locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only, we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV) prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus, we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN) and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences, flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed, three contained the HSV1-TK transgene at the OCT4 locus, but they were not sensitive to GCV. The other six clones were GCV-sensitive, but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days, indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed.

Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells

We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.

In vitro generation of hiPSC-derived megakaryocytes and platelets from a patient with Glanzmann thrombasthenia

Glanzmann Thrombasthenia (GT) is an autosomal recessive platelet disorder characterized by impaired fibrinogen binding due to mutations in the integrin receptor GPIIbIIIa. GT has been studied using animal models, heterologous expression systems and patient-derived platelets. Patient-specific human induced pluripotent stem cells (hiPSC) offer new approaches for modeling blood disorders in vitro by differentiating hiPSC-derived haematopoietic stem cells (HSCs) into myeloid and lymphoid lineages. We have generated patient-specific hiPSC of an 18 year old female carrying two novel compound heterozygous GPIIb mutations. HiPSC clones were in vitro differentiated into HSCs, megakaryocytes (MKs) and platelets using a specific protocol. As expected MKs and platelets from both control and GT-hiPSC showed normal morphology in haemacolour stains and similar expression of CD42b (GPIb). No surface or intracellular expression of CD41 protein (GPIIb) was detected in MKs and platelets from GT-hiPSC. Upon activation with Thromboxan A and ADP, peripheral blood-derived and GT-hiPSC-derived platelets showed reduced PAC1 binding, surface spreading and adherence on fibrinogen, resembling the in vitro phenotype of GT. We demonstrate that GT-hiPSC-derived MKs and platelets recapitulate the disease phenotype in vitro. ![Figure][1] ESC_Glanzmann This model offers the ability to study pathophysiology in MKs which are difficult to obtain from patients with bleeding disorders. In the future it can be a useful tool to elucidate the pathomechanism of disease-causing mutations in platelet receptors and thereby facilitating the in vitro evaluation of new therapeutic anti-platelet agents. After gene correction it might even open the road for gene therapy and transplantation of corrected hIPSC derived HSCs. [1]: pending:yes

Feeder-Free Generation and Long-Term Culture of Human Induced Pluripotent Stem Cells Using Pericellular Matrix of Decidua Derived Mesenchymal Cells

Human ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cells’ pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs.

A method for efficiently generating neurospheres from human-induced pluripotent stem cells using microsphere arrays

In vitro, human neural stem cells can be selectively expanded from fetal or adult neural tissues as neurospheres consisting of immature neural progenitor cells. Access to human neural tissues is limited, making it difficult to propagate and use primary neural stem or progenitor cells (NSPCs) from human neural tissues (hN-NSPCs). It was recently demonstrated that hN-NSPCs can be differentiated from either human embryonic stem cells (hESC-NSPCs) or human-induced pluripotent stem cells (hiPSC-NSPCs), and that hESC-NSPCs and hiPSC-NSPCs are adaptable, powerful substitutes for hN-NSPCs in both regenerative medicine and pharmacological or neurotoxicological assays. We here describe a new protocol to generate neurospheres consisting of hiPSC-NSPCs using microsphere arrays, the surface of which is modified with polyethylene glycol to render it nonadhesive to cells. Primary hiPSCs treated with noggin formed neurospheres on the microsphere arrays and could be stably propagated as free-floating spheroids. The hiPSC-NSPCs proliferating in these neurospheres were almost identical in phenotype to hN-NSPCs, in both cell-surface marker expression and their ability to differentiate into neuronal cells, although gene expression profiles showed that the hiPSC-NSPCs had higher neural and lower glial gene expression, along with mid-hindbrain-like regional specificity. This convenient propagation protocol can be used to evaluate the neurosphere-forming efficiency of hiPSC clones. This method will support the generation of neurospheres from hESCs and hiPSCs and contribute to the use of hESC-NSPCs and hiPSC-NSPCs in research.

Ultrastructural Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Long-Term Culture

In the short- to mid-term, cardiomyocytes generated from human-induced pluripotent stem cells (hiPSC-CMs) have been reported to be less mature than those of adult hearts. However, the maturation process in a long-term culture remains unknown. A hiPSC clone generated from a healthy control was differentiated into CMs through embryoid body (EB) formation. The ultrastructural characteristics and gene expressions of spontaneously contracting EBs were analyzed through 1-year of culture after cardiac differentiation was initiated. The 14-day-old EBs contained a low number of myofibrils, which lacked alignment, and immature high-density Z-bands lacking A-, H-, I-, and M-bands. Through the long-term culture up to 180 days, the myofibrils became more tightly packed and formed parallel arrays accompanied by the appearance of mature Z-, A-, H-, and I-bands, but not M-bands. Notably, M-bands were finally detected in 360-day-old EBs. The expression levels of the M-band-specific genes in hiPSC-CMs remained lower in comparison with those in the adult heart. Immunocytochemistry indicated increasing number of MLC2v-positive/MLC2a-negative cells with decreasing number of MLC2v/MLC2a double-positive cells, indicating maturing of ventricular-type CMs. The structural maturation process of hiPSC-CMs through 1-year of culture revealed ultrastructural sarcomeric changes accompanied by delayed formation of M-bands. Our study provides new insight into the maturation process of hiPSC-CMs.

Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells

Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation.

A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs

Human induced pluripotent stem cells (hiPSCs) hold enormous potential, however several obstacles impede their translation to industrial and clinical applications. Here we describe a platform to efficiently generate, characterize and maintain single cell and feeder-free (FF) cultured hiPSCs by means of a small molecule cocktail media additive. Using this strategy we have developed an effective multiplex sorting and high-throughput selection platform where individual clonal hiPSC lines are readily obtained from a pool of candidate clones, expanded and thoroughly characterized. By promoting survival and self-renewal, the selected hiPSC clones can be rapidly expanded over multiple FF, single-cell passages while maintaining their pluripotency and genomic stability as demonstrated by trilineage differentiation, karyotype and copy number variation analysis. This study provides a robust platform that increases efficiency, throughput, scale and quality of hiPSC generation and facilitates the industrial and clinical use of iPSC technology.

The Effect of Sperm Concentration in the Ejaculate on Morphological Traits of Bull Spermatozoa

Experiments were performed on 75 ejaculates obtained from 19 bulls representing different cattle breeds used at the Masovian Centre for Animal Breeding and Reproduction in Łowicz. Fresh ejaculates were measured in respect to their volume and sperm count in the ejaculates was determined. The ejaculates were classified based on the criterion of sperm concentration and divided into five groups. Sperm morphometric measurements were taken from each bull and assessment of semen morphology was done on the basis of examination under a microscope using preparations made from fresh ejaculates. For each slide, morphometric measurements were taken of 15 randomly selected spermatozoa characterised by normal morphology and well visible under the microscope. Additionally, in each preparation morphometry of 500 spermatozoa was evaluated, numbers of spermatozoa with normal morphology and morphological abnormalities were recorded and these were categorized into spermatozoa with major and minor defects. An insignificant correlation was observed between the sperm concentration in the ejaculate and morphological traits, dimensions and shapes of bull spermatozoa. The less concentrated ejaculates contained spermatozoa with a slightly larger head circumference and a more elongated head shape in comparison with the spermatozoa in the more concentrated ejaculates. The highest frequency of morphologically malformed spermatozoa, both in the case of primary and secondary alterations, was observed in ejaculates with sperm concentration of no more than 1000 x 10(3)/mm3.