Mouse iPSCs Research Articles

Passage number is a major contributor to genomic structural variations in mouse iPSCs.

Emergence of genomic instability is a practical issue in preparing neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs). However, it is still not fully understood what the origins and mechanisms for formation are for the genomic alternations observed. Here, we studied the extent of genomic variation on the scale of individual cells originating from the same animal. We used mouse NSCs grown from embryonic cells and iPSCs generated from embryonic brain cells, B cells or fibroblasts, and performed comparative analysis with cultures of fibroblasts from the same mouse. In the first passage of these cell lines, aneuploidies were only observed for chromosomes 6, 11, 12, 19, and Y, which is overall at a rate lower than previously reported; de novo copy number variations (CNVs) were observed in 4.3% of neural iPSCs, 29% of B cell iPSCs, 10% of fibroblast iPSCs, and 1.3% of neurospheres. In contrast, propagation of these first passage cells to a later passage induced additional aneuploidies and CNVs. Breakpoint sequencing analysis suggested that the majority of the detected CNVs arose by replicative mechanisms. Interestingly, we detected identical de novo CNVs in different single cell colonies that appeared to have arisen independently from each other, which suggests a novel CNV formation mechanism in these cells. Our findings provide insights into mechanisms of CNV formation during reprogramming and suggest that replicative mechanisms for CNV formation accompany mitotic divisions.

A Cell Engineering Strategy to Enhance the Safety of Stem Cell Therapies

The long-term risk of malignancy associated with stem cell therapies is a significant concern in the clinical application of this exciting technology. We report a cancer-selective strategy to enhance the safety of stem cell therapies. Briefly, using a cell engineering approach, we show that aggressive cancers derived from human or murine induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are strikingly sensitive to temporary MYC blockade. On the other hand, differentiated tissues derived from human or mouse iPSCs can readily tolerate temporary MYC inactivation. In cancer cells, endogenous MYC is required to maintain the metabolic and epigenetic functions of the embryonic and cancer-specific pyruvate kinase M2 isoform (PKM2). In summary, our results implicate PKM2 in cancer's increased MYC dependence and indicate dominant MYC inhibition as a cancer-selective fail-safe for stem cell therapies.

Histone Variant H2A.X Deposition Pattern Serves as a Functional Epigenetic Mark for Distinguishing the Developmental Potentials of iPSCs

For future application of induced pluripotent stem cell (iPSC) technology, the ability to assess the overall quality of iPSC clones will be an important issue. Here we show that the histone variant H2A.X is a functional marker that can distinguish the developmental potentials of mouse iPSC lines. We found that H2A.X is specifically targeted to and negatively regulates extraembryonic lineage gene expression in embryonic stem cells (ESCs) and prevents trophectoderm lineage differentiation. ESC-specific H2A.X deposition patterns are faithfully recapitulated in iPSCs that support the development of "all-iPS" animals via tetraploid complementation, the most stringent test available of iPSC quality. In contrast, iPSCs that fail to support all-iPS embryonic development show aberrant H2A.X deposition, upregulation of extraembryonic lineage genes, and a predisposition to extraembryonic differentiation. Thus, our work has highlighted an epigenetic mechanism for maintaining cell lineage commitment in ESCs and iPSCs that can be used to distinguish the quality of iPSC lines.

Study on the induced differentiation of induced pluripotent stem cells into cochlear hair cell-like cells and spiral ganglion neuron-like cells in vitro

In this study, we investigated the potential of mouse induced pluripotent stem cells (iPSC) for use as a source of transplants for the restoration of auditory hair cells and spiral ganglion neurons. We co-cultured the mouse iPSC with the cells of the cochlear organ of Corti or the modiolus in vitro. The cochlear organ of Corti (which contains cochlear hair cells) and the modiolus (which contains auditory spiral ganglion neurons) were obtained from postnatal day 3 (P3) CD-1 ICR mice. After 18 days of coculture with the cells of newborn mouse cochleae. The expressions of hair cell markers (Myosin VIIa, Math1, Calretinin, Espin) and Spiral ganglion neuron markers [Nestin, Neurofilament-M, β-III Tubulin, Vesicular glutamate transporter 1(VGluT1)] were detected by immunocytochemical analysis. Immunocytochemical analysis results indicated that the differentiated iPSC expressed auditory hair cell markers (MyosinVIIa,Math1, Calretinin, Espin ) and spiral ganglion markers (Nestin, Neurofilament-M,β-III Tubulin,VGluT1). Mouse iPSC in virto cultured could successfully be induced to differentiate into hair cell-like cells and spiral ganglion-like cells with hair cell and spiral ganglion molecular markers.

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells.

In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.

Insulin‐Like Growth Factor Binding Protein 4 Enhances Cardiomyocytes Induction in Murine‐Induced Pluripotent Stem Cells

Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes cardiomyocytes induction of iPSCs is unclear. In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. We observed that IGFBP4 treatment at late stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac-specific transcription factor), α-MHC, α-actinin, and Troponin I (cardiac-specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that α-MHC, TNNT2 and connexin 43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs, but it significantly promoted the proliferation of cardiomyocytes from iPSCs characterized by higher ratio EdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibited β-catenin expression in cytosol of EBs at late stage during differentiation of iPSCs. Knockdown of β-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhanced cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocytes differentiation of iPSCs has relationship with β-catenin signaling pathway. In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through inhibiting β-catenin signaling.

Low oxygen tension enhances the generation of lung progenitor cells from mouse embryonic and induced pluripotent stem cells.

Whole‐organ decellularization technology has emerged as a new alternative for the fabrication of bioartificial lungs. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are potentially useful for recellularization since they can be directed to express phenotypic marker genes of lung epithelial cells. Normal pulmonary development takes place in a low oxygen environment ranging from 1 to 5%. By contrast, in vitro ESC and iPSC differentiation protocols are usually carried out at room‐air oxygen tension. Here, we sought to determine the role played by oxygen tension on the derivation of Nkx2.1+ lung/thyroid progenitor cells from mouse ESC and iPSC. A step‐wise differentiation protocol was used to generate Nkx2.1+ lung/thyroid progenitors under 20% and 5% oxygen tension. On day 12, gene expression analysis revealed that Nkx2.1 and Foxa2 (endodermal and early lung epithelial cell marker) were significantly upregulated at 5% oxygen tension in ESC and iPSC differentiated cultures compared to 20% oxygen conditions. In addition, quantification of Foxa2+Nkx2.1+Pax8‐ cells corresponding to the lung field, with exclusion of the potential thyroid fate identified by Pax8 expression, confirmed that the low physiologic oxygen tension exerted a significant positive effect on early pulmonary differentiation of ESC and iPSC. In conclusion, we found that 5% oxygen tension enhanced the derivation of lung progenitors from mouse ESC and iPSC compared to 20% room‐air oxygen tension.

Deletion of Alox5 gene decreases osteogenic differentiation but increases adipogenic differentiation of mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have great potential in bone tissue engineering to repair large bone defects. Before their clinical application, investigations are needed to discover the genes and osteoconductive scaffolds that influence their differentiation toward an osteogenic lineage. Alox5 plays controversial and complex roles in the regulation of bone and fat metabolism. To detect the effect of Alox5 on osteogenic and adipogenic differentiation of iPSCs, both Alox5 knockout mouse iPSCs (Alox5-KO-iPSCs) and wild-type mouse iPSCs (Wild-iPSCs) were developed. The mRNA levels of many osteogenic markers in Alox5-KO-iPSCs were significantly reduced, while many adipogenic markers were enhanced. Furthermore, when implanted in rat cranial critical-sized defects with collagen/chitosan/hydroxyapatite scaffolds (CCHS), Alox5-KO-iPSCs produced significantly less new bone than Wild-iPSCs and both cell-scaffold groups had no tumor formation. There was a significant difference in the expression of Cox2 during the osteogenic and adipogenic differentiation between the two kinds of iPSCs in vitro. In conclusion, firstly, Alox5 knockout reduced the osteogenic but increased the adipogenic differentiation potential of mouse iPSCs. These disorders might be related to the change of Cox2 expression. Secondly, combined with iPSCs, CCHS can serve as a potential substrate to repair critical-sized bony defects. However, more studies are required to confirm the mechanisms through which Alox5 affects the osteogenic and adipogenic abilities of iPSCs in vivo and the effect of Cox2 inhibition in this system.

Repression of SIRT1 promotes the differentiation of mouse induced pluripotent stem cells into neural stem cells.

The use of transplanting functional neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) has increased for the treatment of brain diseases. As such, it is important to understand the molecular mechanisms that promote NSCs differentiation of iPSCs for future NSC-based therapies. Sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase, has attracted significant attention over the past decade due to its prominent role in processes including organ development, longevity, and cancer. However, it remains unclear whether SIRT1 plays a role in the differentiation of mouse iPSCs toward NSCs. In this study, we produced NSCs from mouse iPSCs using serum-free medium supplemented with retinoic acid. We then assessed changes in the expression of SIRT1 and microRNA-34a, which regulates SIRT1 expression. Moreover, we used a SIRT1 inhibitor to investigate the role of SIRT1 in NSCs differentiation of iPSCs. Data revealed that the expression of SIRT1 decreased, whereas miRNAs-34a increased, during this process. In addition, the inhibition of SIRT1 enhanced the generation of NSCs and mature neurocytes. This suggests that SIRT1 negatively regulated the differentiation of mouse iPSCs into NSCs, and that this process may be regulated by miRNA-34a.

Fine-Tuning of iPSC Derivation by an Inducible Reprogramming System at the Protein Level

SummaryInduced pluripotent stem cells (iPSCs) generated from somatic cells by ectopic expression of reprogramming factors, e.g., POU5F1 (OCT4), KLF4, and SOX2, have great potential for regenerative medicine. However, before they can be used in a clinical setting, the mechanism of reprogramming needs to be better understood. Here, by engineering reprogramming factors to a destabilizing protein domain, we achieved inducible generation of mouse and pig iPSCs. Stability of the fusion protein was precisely regulated by the addition of the cell-permeable small molecule trimethoprim (TMP) in a dose-dependent manner. With these tools, we found that during the early and middle stages of reprogramming, exogenous OCT4 or KLF4 could be omitted, whereas exogenous SOX2 expression at early and middle stages was required for successful reprogramming. Our TMP reprogramming system is useful for defining the stoichiometry and temporal requirements of transcription factors for reprogramming.

Understanding the roadmaps to induced pluripotency.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of transcription factors Oct4, Sox2, Klf4 and cMyc. Recent advancements have shown that small-molecule compounds can induce pluripotency, indicating that cell fate can be regulated by direct manipulation of intrinsic cell signaling pathways, thereby innovating our current understanding of reprogramming. The fact that lineage specifiers can induce pluripotency suggests that the pluripotent state is a fine balance between competing differentiation forces. Dissection of pluripotent roadmaps indicates that reprogramming is a process of reverse development, involving a series of complicated and distinct reprogramming stages. Evidence from mouse iPSC transplantation studies demonstrated that some certain but not all cells derived from iPSCs are immunogenic. These studies provide new ways to minimize reprogramming-induced abnormalities and maximize reprogramming efficiency to facilitate clinical development and use of iPSCs.

Differentiation of mouse induced pluripotent stem cells into alveolar epithelial cells in vitro for use in vivo.

Alveolar epithelial cells (AECs) differentiated from induced pluripotent stem cells (iPSCs) represent new opportunities in lung tissue engineering and cell therapy. In this study, we modified a two-step protocol for embryonic stem cells that resulted in a yield of ∼9% surfactant protein C (SPC)(+) alveolar epithelial type II (AEC II) cells from mouse iPSCs in a 12-day period. The differentiated iPSCs showed morphological characteristics similar to those of AEC II cells. When differentiated iPSCs were seeded and cultured in a decellularized mouse lung scaffold, the cells reformed an alveolar structure and expressed SPC or T1α protein (markers of AEC II or AEC I cells, respectively). Finally, the differentiated iPSCs were instilled intratracheally into a bleomycin-induced mouse acute lung injury model. The transplanted cells integrated into the lung alveolar structure and expressed SPC and T1α. Significantly reduced lung inflammation and decreased collagen deposition were observed following differentiated iPSC transplantation. In conclusion, we report a simple and rapid protocol for in vitro differentiation of mouse iPSCs into AECs. Differentiated iPSCs show potential for regenerating three-dimensional alveolar lung structure and can be used to abrogate lung injury.

Differentiation of the mouse induced pluripotent stem cells to male germ cells through retinoic acid induction and in the reconstituted seminiferous tubules

Objective To investigate the differentiation potential of induced pluripotent stem cells (iPSCs) into male germ cells through retinoic acid (RA) induction in vitro and ectopic transplantation in vivo.Methods Mouse iPSCs were first transferred to bacterial plates to form embryoid body (EB).After 5days,EB was treated by 2 μmol/L RA.After 10 days of cuhure,iPSCs-derived cells were dissociated from EB.The real-time PCR and immunocytochemical assay were used to detect the expression of related gene and protein.Then,they were injected into the male nude mice with testicular cells from neonatal mice.The grafts were obtained for further HE and immunohistochemical staining on 4,6,8,12 weeks after injection.Results After EB formation and RA induction,the expressions of Stra8,Odf2,Act,and Prm1 were upregulated in induction group,which were 4.50± 1.02,1.48±0.04,10.52±0.25,323.28±25.64 times as control group,respectively.On the contrary,the expressions of Oct-4,Dppa3,Piwil2,Tex14,and Scp3 were down-regulated in induction group,which were 0.56±0.02,0.11±0.01,0.50±0.01,0.53±0.14,0.16±0.00times as control group,respectively.Meanwhile,RA-treated EBs also expressed male germ cell markers VASA and SCP3.HE staining showed that the seminiferous tubules could be reconstituted after injecting the iPSCs-derived cells and testicular cells from neonatal mice.Immunofluorescence analysis revealed co-localization of EGFP-positive and VASA-positive cells in the reconstituted tubules.Conclusions Mouse iPSCs could differentiate into male germ cells with EB formation and RA induction.The iPSCs-derived germ cells could settle in the basement membrane of reconstituted tubules,which indicates reconstituted seminiferous tubules may provide a functional niche for exogenous iPSCs-derived male germ cells. Key words: Induced pluripotent stem cells; Embryoid body; Retinoic acid; Ectopic reconstitution; Male germ cells

Detailed Analysis of the Genetic and Epigenetic Signatures of iPSC-Derived Mesodiencephalic Dopaminergic Neurons

SummaryInduced pluripotent stem cells (iPSCs) hold great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson’s disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterizations of such neurons are lacking. The goal of this study was to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS purified mdDA (Pitx3Gfp/+) neurons derived from mouse iPSCs and primary mdDA (Pitx3Gfp/+) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopted characteristics of their in vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed because they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in vitro disease modeling or cell-based therapy.

Cardiomyocyte differentiation of mouse iPSCs regulated by polypeptide multilayer films (1180.21)

Polyelectrolyte multilayer film is an emerging method for substrate coating to modulate cellular behaviors. This material allows for modulation of thickness, stiffness, and adhesiveness. In this study, mouse induced pluripotent stem cells (iPSCs) were cultured on polypeptide multilayer films built up by the alternate deposition of cationic poly‐L‐lysine and anionic poly‐L‐glutamic acid, cross‐linked with EDC/sulfo‐NHS, and coated with gelatin. The characteristics of film thickness, roughness, and shear modulus were measured by AFM and QCM. Cardiomyocyte differentiation was evaluated by RT‐PCR analysis and immunostaining of cardiac markers, and cardiomyocyte beating was monitored by polypeptide multilayer‐coated electrodes using impedance sensing technique. The results showed that mouse iPSC colonies demonstrated better cell spreading and differentiation on even number layer of films, where the top layers are poly‐L‐glutamic acid films. In addition, the cardiomyocyte differentiation of mouse iPSCs showed a significant increase in the differentiation rate when higher concentration of EDC/sulfo‐NHS cross‐linkers was used to form the films. Our observations indicate a possible way to promote iPSCs toward cardiomyocyte differentiation on polypeptide multilayer films by changing the outermost polypeptide layer, the concentration of cross‐linkers, and the number of layers in the film.Grant Funding Source: Supported by the National Science Council in Taiwan

Transient folate deprivation in combination with small-molecule compounds facilitates the generation of somatic cell-derived pluripotent stem cells in mice

Induced pluripotent stem cells (iPSCs) can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical research. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel approach to generating iPSCs. In this study, a new combination of small-molecule compounds (SMs) (sodium butyrate, A-83-01, CHIR99021, Y-27632) under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts (MEFs) in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs. The resulting cell lines resembled mouse embryonic stem (ES) cells with respect to proliferation rate, morphology, pluripotency-associated markers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcinogenicity and the use of exogenous reprogramming factors.

Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production

Induced pluripotent stem cells (iPSCs) are expected to be an ideal cell source for biomedical applications, but such applications usually require a large number of cells. Suspension culture of iPSC aggregates can offer high cell yields but sometimes results in excess aggregation or cell death by shear stress. Hydrogel-based microencapsulation can solve such problems observed in Suspension culture, but there is no systematic evaluation of the possible capsule formulations. In addition, their biological effects on entrapped cells are still poorly studied so far. We, therefore, immobilized mouse iPSCs in three different types of calcium-alginate (Alg-Ca) hydrogel-based microcapsules; (i) Alg-Ca capsules without further treatment (Naked), (ii) Alg-Ca capsules with poly-l-lysine (PLL) coating (Coated), and (iii) Alg-PLL membrane capsules with liquid cores (Hollow). After 10 days of culture within the medium containing serum and leukemia inhibitory factor, we obtained good cellular expansions (10-13-fold) in Coated and Hollow capsules that were similar to Suspension culture. However, 32 ± 9% of cellular leakage and lower cell yield (about threefold) were observed in Naked capsules. This was not observed in Coated and Hollow capsules. In addition, immunostaining and quantitative RT-PCR showed that the formation of primitive endodermal layers was suppressed in Coated capsules contrary to all other formulations. This agenesis of primitive endoderm layers in Coated capsules is likely to be the main cause of the significantly better pluripotency maintenance in hydrogel-based encapsulation culture. These results are helpful in further optimizing hydrogel-based iPSC culture, which can maintain better local cellular environments and be compatible with mass culture.

PT356 VCP746, a novel A1/A2 adenosine receptor agonist, reduces hypertrophy in cardiac myocytes and collagen synthesis in cardiac fibroblasts and renal mesangial cells

Introduction: The establishment of induced pluripotent stem cells (iPSCs) potentially provides a unique resource for generating patient specific cardiomyocytes to for cardiac repair. However, the poor efficiency and yield of cardiomyocytes currently limits the application of this technology. Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes iPSCs into cardiomyocytes is unclear. Objectives: In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. Methods: For differentiation, iPSCs were trypsinized and EBs were generated by the threedimensional hanging-drop method. EBs were grown in hanging drops for three days (day 0 to day 3), each drop initially consisted of 1000 cells in 30 mL of differentiation medium (growth medium without LIF). IGFBP4 (Sino Biological Inc.) was added at day 3-8,day 3-6 and day 6-8 period under 1 mg/ mL to determine the effective time-window. Results: We observed that IGFBP4 treatment at late-stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac specific transcription factor), a-MHC, a-actinin and Troponin I (cardiac specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that a-MHC, TNNT2 and connex43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs. But it significantly promoted the proliferation of cardiomycytes from iPSCs chracterized by higher ratio BrdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibted b-catenin expression in cytosol of EBs at late-stage during differentiation of iPSCs. Knockdown of b-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhancing cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocyte differentiation of iPSCs has relationship with b-catenin signaling pathway. Conclusion: In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through b-catenin signaling. Disclosure of Interest: None Declared

PT353 Identification of miRNA networks in pathological cardiac remodelling-New therapeutic targets

Introduction: The establishment of induced pluripotent stem cells (iPSCs) potentially provides a unique resource for generating patient specific cardiomyocytes to for cardiac repair. However, the poor efficiency and yield of cardiomyocytes currently limits the application of this technology. Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes iPSCs into cardiomyocytes is unclear. Objectives: In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. Methods: For differentiation, iPSCs were trypsinized and EBs were generated by the threedimensional hanging-drop method. EBs were grown in hanging drops for three days (day 0 to day 3), each drop initially consisted of 1000 cells in 30 mL of differentiation medium (growth medium without LIF). IGFBP4 (Sino Biological Inc.) was added at day 3-8,day 3-6 and day 6-8 period under 1 mg/ mL to determine the effective time-window. Results: We observed that IGFBP4 treatment at late-stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac specific transcription factor), a-MHC, a-actinin and Troponin I (cardiac specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that a-MHC, TNNT2 and connex43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs. But it significantly promoted the proliferation of cardiomycytes from iPSCs chracterized by higher ratio BrdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibted b-catenin expression in cytosol of EBs at late-stage during differentiation of iPSCs. Knockdown of b-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhancing cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocyte differentiation of iPSCs has relationship with b-catenin signaling pathway. Conclusion: In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through b-catenin signaling. Disclosure of Interest: None Declared

Genome-wide CNV analysis in mouse induced pluripotent stem cells reveals dosage effect of pluripotent factors on genome integrity

BackgroundInduced pluripotent stem cells (iPSCs) derived from somatic cells have enormous potential for clinical applications. Notably, it was recently reported that reprogramming from somatic cells to iPSCs can induce genomic copy number variation (CNV), which is one of the major genetic causes of human diseases. However it was unclear if this genome instability is dependent on reprogramming methods and/or the genetic background of donor cells. Furthermore, genome-wide CNV analysis is technically challenging and CNV data need to be interpreted with care.ResultsIn order to carefully investigate the possible CNV instability during somatic reprogramming, we performed genome-wide CNV analyses with 41 mouse iPSC lines generated from the same parental donor; therefore, the donor’s genetic background can be controlled. Different reprogramming factor combinations and dosages were used for investigating potential method-dependent effects on genome integrity. We detected 63 iPSC CNVs using high-resolution comparative genomic hybridization. Intriguingly, CNV rates were negatively associated with the dosages of classic factor(s). Furthermore, the use of high-performance engineered factors led to less CNVs than the classic factor(s) of the same dosage.ConclusionOur observations suggest that sufficient reprogramming force can protect the genome from CNV instability during the reprogramming process.