Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy.

BACKGROUND: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). METHODS: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. RESULTS: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. CONCLUSIONS: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.

Accumulation of instability in serial differentiation and reprogramming of parthenogenetic human cells.

Human leukocyte antigen-homozygous parthenogenetic stem cells (pSC) could provide a source of progenitors for regenerative medicine, lowering the need for immune suppression in patients. However, the high level of homozygosis and the lack of a paternal genome might pose a safety challenge for their therapeutic use, and no study so far has evaluated the spread and significance of gene expression changes across serial potency changes in these cells. We performed serial rounds of differentiation and reprogramming to assess pSC gene expression stability, likely of epigenetic source. We first derived pSC from activated MII oocytes, and differentiated them to parthenogenetic mesenchymal stem cells (pMSC). We then proceeded to induce pluripotency in pMSC by over expression of the four transcription factors Oct4, Sox2, Klf4 and c-Myc. pMSC-derived iPS (piPS) were further differentiated into secondary pMSC (pMSC-II). At every potency change, we characterized the obtained lines both molecularly and by functional differentiation, and performed an extensive genome-wide expression study by microarray analysis. Although overall gene expression of parthenogenetic cells resembled that of potency-matched biparental lines, significantly broader changes were brought about upon secondary differentiation of piPS to pMSC-II compared with matched biparental controls; our results highlight the effect of the interplay of epigenetic reprogramming on a monoparental background, as well as the importance of heterozygosis and biparental imprinting for stable epigenetic reprogramming.

Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development.

The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.

Effect of the combination of different depigmenting agents in vitro.

Melanin plays a key role in our skin, protecting us against ultraviolet radiation, but there are situations in which its anomalous accumulation can lead to either aesthetic problems or diseases like melasma. For this reason, it is important to find agents that are able to decrease the skin pigmentation. It has been demonstrated that the melanin synthesis pathway can be inhibited at different levels by different mechanisms of action. The aim of this project is to combine some of these agents with different mechanisms of action on this pathway in order to find synergistic effects in the inhibition of tyrosinase and melanin synthesis. Kojic acid + α-lipoic acid combination are the only ones that have shown a synergistic effect over mushroom tyrosinase. However, this effect is not seen in melanin synthesis inhibition, although this combination is the most effective one. A potentiation effect is seen in arbutin + α-lipoic acid and kojic acid + azelaic acid combination. Kojic acid and α-lipoic acid combination might prove a good approach as treatment for hyperpigmentation disorders.

The pluripotency state of human embryonic stem cells derived from single blastomeres of eight-cell embryos.

Human embryonic stem cells (hESCs) derived from blastocyst stage embryos present a primed state of pluripotency, whereas mouse ESCs (mESCs) display naïve pluripotency. Their unique characteristics make naïve hESCs more suitable for particular applications in biomedical research. This work aimed to derive hESCs from single blastomeres and determine their pluripotency state, which is currently unclear. We derived hESC lines from single blastomeres of 8-cell embryos and from whole blastocysts, and analysed several naïve pluripotency indicators, their transcriptomic profile and their trilineage differentiation potential. No significant differences were observed between blastomere-derived hESCs (bm-hESCs) and blastocyst-derived hESCs (bc-hESCs) for most naïve pluripotency indicators, including TFE3 localization, mitochondrial activity, and global DNA methylation and hydroxymethylation, nor for their trilineage differentiation potential. Nevertheless, bm-hESCs showed an increased single-cell clonogenicity and a higher expression of naïve pluripotency markers at early passages than bc-hESCs. Furthermore, RNA-seq revealed that bc-hESCs overexpressed a set of genes related to the post-implantational epiblast. Altogether, these results suggest that bm-hESCs, although displaying primed pluripotency, would be slightly closer to the naïve end of the pluripotency continuum than bc-hESCs.

Generation of the induced pluripotent stem cell line ESi108-A from a familial atrial fibrillation patient.

Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.

Generation of an induced pluripotent stem cell line (ESi107-A) from a transthyretin amyloid cardiomyopathy (ATTR-CM) patient carrying a p.Ser43Asn mutation in the TTR gene.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery.

Vitrified blastocysts from Preimplantation Genetic Diagnosis (PGD) as a source for human Embryonic Stem Cell (hESC) derivation.

Embryos diagnosed as abnormal in Preimplantation Genetic Diagnosis (PGD) cycles are useful for the establishment of human Embryonic Stem Cells (hESC) lines with genetic disorders. These lines can be helpful for drug screening and for the development of new treatments. Vitrification has proved to be an efficient method to preserve human blastocysts. One hundred and three abnormal or undiagnosed vitrified blastocysts from the PGD programme at Institut Universitari Dexeus were donated for human embryonic stem cell derivation. The overall survival rate after warming was 70.6 %. Our results showed better survival rates when blastocysts have not started the hatching process (initial/expanded 87.8 %, hatching 68.3 % and hatched 27.3 %). Thirty-five blastocysts and 12 partially surviving embryos were seeded. One hESC line with the multiple exostoses type 2 paternal mutation was obtained.

Generation of four induced pluripotent stem cell lines from a family harboring a single nucleotide variant in SCN5A.

Patient-derived induced pluripotent stem cells (iPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of three family members carrying a single nucleotide variant (SNV) in SCN5A, which encodes the cardiac-type sodium channel, and of a related healthy control. The SNV SCN5A_c.4573G > A had been previously identified in a Brugada Syndrome patient. The resulting iPS cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express pluripotency surface markers and genes. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.

Generation of an induced pluripotent stem cell line from a healthy Caucasian male.

The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC.

Pluripotent stem cell regulation in Spain and the Spanish National Stem Cell Bank.

The Spanish National Stem Cell Bank (Banco Nacional de Líneas Celulares, BNLC) was established in 2006 thanks to a change in the legislative framework in Spain. The Law 14/2006 updated the previous Assisted Reproduction Techniques Law (Law 45/2003) allowing the use of the surplus frozen embryos following IVF for research. The BNLC has a network structure with 3 nodes: the Regenerative Medicine Program (IDIBELL), the Principe Felipe Research Center (CIPF) in Valencia and the Andalusian Public Health System Biobank (SSPA Biobank) in Granada. The aim of the BNLC is to guarantee throughout the national territory the availability of human stem cell lines for biomedical research. At present time, there are 40 human embryonic stem cell lines (hESC) and 171 human induced pluripotent stem cell lines (hiPSC) registered in the BNLC. These lines are fully characterized and available in the context of research projects approved by the Technical Committee of the BNLC.

EBCOG position statement: ethics of stem cell research.

Over the past 10 years' significant research developments have taken place on human pluripotent stem cells and human embryonic stem cells to exploit the future potential in gene therapy and other focused treatments. There remains concerns around ethics of research and the fate of the human embryo used in such studies. European Board and College of Obstetrics and Gynaecology urge upon all scientists and the research bodies to adhere to the highest ethical principles of confidentiality and their actions should meet the criteria as set out by the international society for stem cell research.

Access to stem cell data and registration of pluripotent cell lines: The Human Pluripotent Stem Cell Registry (hPSCreg).

The value of human pluripotent stem cells (hPSC) in regenerative medicine has yet to reach its full potential. The road from basic research tool to clinically validated PSC-derived cell therapy products is a long and winding one, leading researchers, clinicians, industry and regulators alike into undiscovered territory. All stakeholders must work together to ensure the development of safe and effective cell therapies. Similarly, utilization of hPSC in meaningful and controlled disease modeling and drug screening applications requires information on the quality and suitability of the applied cell lines. Central to these common goals is the complete documentation of hPSC data, including the ethical provenance of the source material, the hPSC line derivation, culture conditions and genetic constitution of the lines. Data surrounding hPSC is scattered amongst diverse sources, including publications, supplemental data, researcher lab books, accredited lab reports, certificates of analyses and public data repositories. Not all of these data sources are publicly accessible nor associated with metadata nor stored in a standard manner, such that data can be easily found and retrieved. The Human Pluripotent Stem Cell Registry (hPSCreg; https://hpscreg.eu/) was started in 2007 to impart provenance and transparency towards hPSC research by registering and collecting standard properties of hPSC lines. In this chapter, we present a short primer on the history of stem cell-based products, summarize the ethical and regulatory issues introduced in the course of working with hPSC-derived products and their associated data, and finally present the Human Pluripotent Stem Cell Registry as a valuable resource for all stakeholders in therapies and disease modeling based on hPSC-derived cells.

Generation of integration-free induced pluripotent stem cell lines derived from two patients with X-linked Alport syndrome (XLAS).

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing.

Integration-free induced pluripotent stem cells derived from a patient with autosomal recessive Alport syndrome (ARAS).

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing.

Outcome of intracytoplasmic sperm injection in relation to the meiotic pattern in patients with severe oligoasthenozoospermia.

OBJECTIVE: The aim of the study was to evaluate the intracytoplasmic sperm injection outcome in a selected group of patients with oligoasthenozoospermia in relation to the results obtained from their meiotic analysis. DESIGN: Retrospective clinical study. SETTING: An assisted reproduction service and a university department. PATIENT(S): One hundred thirty-seven men with oligoasthenozoospermia grouped in relation to their meiotic pattern. INTERVENTION(S): Two hundred twenty-four intracytoplasmic sperm injection cycles from 137 men with oligoasthenozoospermia in whom diagnostic meiotic analyses had been performed. MAIN OUTCOME MEASURE(S): Fertilization, pregnancy, implantation, and abortion rates. RESULT(S): There were no significant statistical differences in fertilization, pregnancy, implantation, or abortion rates among the three groups studied. CONCLUSION(S): No statistically significant differences in fertilization, pregnancy, implantation, or abortion rates were found in patients with oligoasthenozoospermia in relation to the meiotic pattern.

Derivation of human embryonic stem cells at the Center of Regenerative Medicine in Barcelona.

We report here the legislative issues related to embryo research and human embryonic stem cell (hESC) research in Spain and the derivation of nine hESC lines at the Center of Regenerative Medicine in Barcelona. You can find the information for obtaining our lines for research purposes at blc@cmrb.eu.

Influence of spermatogenic profile and meiotic abnormalities on reproductive outcome of infertile patients.

Genetic aspects of male infertility and the possible risks of new assisted reproduction and their influence on the development of zygotes and children born after intracytoplasmic sperm injection (ICSI) need further research. These patients have an increased risk of diploidy, and disomies are frequent in their spermatozoa. Meiotic disorders are more common in testicular biopsies of patients with severe oligoasthenozoospermia. For these reasons, a detailed andrological study is absolutely mandatory before accepting a couple with these characteristics into an IVF-ICSI programme. When an andrological patient has plasma FSH values >10 IU/l and/or very low total motile sperm count <1 x 10(6), despite a normal karyotype, they clearly need a testicular biopsy and a meiotic study in order to rule out meiotic arrest or synaptic anomalies. Another important aspect to be considered is the possible benefit of applying preimplantation genetic diagnosis in these cases because they normally have a high percentage of chromosomally abnormal embryos, although in the present study this was not evident. All studies agree on the necessity of conducting follow-up studies in the population of children born after IVF-ICSI. In this way, it will be possible to find out if these infertile patients and their offspring have a higher risk of suffering epigenetic errors and imprinting disorders.

Spermatogenic patterns and early embryo development after intracytoplasmic sperm injection in severe oligoasthenozoospermia.

PURPOSE: Evaluate the influence of different baseline spermatogenic patterns [meiotic pattern (normal or abnormal), sperm concentration (> 1 x 10(6)/mL or < or = 1 x 10(6)/mL), and the combined meiosis-sperm concentration pattern] on early embryo development in severe oligoasthenozoospermia. METHODS: Embryo outcomes (fertilization rate, cleavage rate, and 4-cell stage embryo division rate on day 2) after IVF-ICSI in 75 oligoasthenozoospermia and 79 normozoospermic males. RESULTS: The embryo division rate was significantly lower in oligoasthenozoospermia compared to normozoospermia (50.43% vs. 58.72%, p < 0.01) and in the oligoasthenozoospermia group for meiotic anomalies (43.40%), sperm concentration < or = 1 x 10(6)/mL (44.35%), and the combined pattern < or = 1 x 10(6)/mL with meiotic anomalies (37.17%). Logistic regression analysis showed a synergic effect (OR = 2.00; 95% CI = 1.28-3.12) when the two spermatogenic patterns predictive of slow embryo development [meiotic anomalies (OR = 1.49; 95% CI = 1.03-2.15) and sperm concentration < or = 1 x 10(6)/mL (OR = 1.53; 95% CI = 1.09-2.13)] were present. CONCLUSIONS: The data suggest that the early embryonic developmental capacity is inversely related to the severity of spermatogenic impairment (meiotic anomalies and/or sperm concentration < or = 1 x 10(6)/mL).