Limitations of gene editing assessments in human preimplantation embryos.

Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.

The Outcomes and Quality of Pancreatic Islet Cells Isolated from Surgical Specimens for Research on Diabetes Mellitus.

Isolating a large quantity of high-quality human islets is a prerequisite for diabetes research. Human islets are typically isolated from the pancreases of brain-dead donors, making research difficult due to low availability. Pancreas tissue discarded after surgical resection may be a good alternative source of islet cells. To test this hypothesis, we isolated islets from discarded surgical specimens and evaluated the islet yield and quality as well as islet cell preparations. Eighty-two segmental pancreases were processed using the Ricordi automated method, and islet yield and quality were investigated. The mean age of patients was 54.6, and the cohort included 32 diabetes patients. After purification, partially resected pancreases yielded an average of 59,593 ± 56,651 islet equivalents (IEQs) and 2546 IEQ/g of digested pancreas, with 71.5 ± 21% purity. Multivariate analysis revealed that diabetes (p = 0.0046) and the lobe used (p = 0.0156) significantly altered islet yield. Islets transplanted into diabetic mice displayed good viability and in vitro glucose responses, DNA/RNA quality, mitochondrial function, and glucose control, even though these results were dependent on islet quality. Isolated cells also maintained high viability and function even after cryopreservation. Our findings indicate that pancreatic tissue discarded after surgery can be a valuable source of islets for diabetes research.

Dysfunctional pancreatic cells differentiated from induced pluripotent stem cells with mitochondrial DNA mutations.

Diabetes mellitus (DM) is a serious disease in which blood sugar levels rise abnormally because of failed insulin production or decreased insulin sensitivity. Although many studies are being conducted for the treatment or early diagnosis of DM, it is not fully understood how mitochondrial genome (mtDNA) abnormalities appear in patients with DM. Here, we induced iPSCs from fibroblasts, PBMCs, or pancreatic cells of three patients with type 2 DM (T2D) and three patients with non-diabetes counterpart. The mtDNA mutations were detected randomly without any tendency among tissues or patients. In T2D patients, 62% (21/34) of iPSC clones harbored multiple mtDNA mutations, of which 37% were homoplasmy at the 100% mutation level compared to only 8% in non-diabetes. We next selected iPSC clones that were a wild type or carried mutations and differentiated into pancreatic cells. Oxygen consumption rates were significantly lower in cells carrying mutant mtDNA. Additionally, the mutant cells exhibited decreased production of insulin and reduced secretion of insulin in response to glucose. Overall, the results suggest that screening mtDNA mutations in iPSCs from patients with T2D is an essential step before pancreatic cell differentiation for disease modeling or autologous cell therapy. [BMB Reports 2022; 55(9): 453-458].

Mitochondrial genome mutations and neuronal dysfunction of induced pluripotent stem cells derived from patients with Alzheimer's disease.

OBJECTIVES: Patient-derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC-derived neuronal cells from patients with Alzheimer's disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of ß-amyloid (Aß) in differentiated neuronal cells. MATERIALS AND METHODS: mtDNA mutations were measured and compared among iPSCs and iPSC-derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aß. RESULTS: Most iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aß deposition. CONCLUSION: This study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aß deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.

Artificial Oocyte: Development and Potential Application.

Millions of people around the world suffer from infertility, with the number of infertile couples and individuals increasing every year. Assisted reproductive technologies (ART) have been widely developed in recent years; however, some patients are unable to benefit from these technologies due to their lack of functional germ cells. Therefore, the development of alternative methods seems necessary. One of these methods is to create artificial oocytes. Oocytes can be generated in vitro from the ovary, fetal gonad, germline stem cells (GSCs), ovarian stem cells, or pluripotent stem cells (PSCs). This approach has raised new hopes in both basic research and medical applications. In this article, we looked at the principle of oocyte development, the landmark studies that enhanced our understanding of the cellular and molecular mechanisms that govern oogenesis in vivo, as well as the mechanisms underlying in vitro generation of functional oocytes from different sources of mouse and human stem cells. In addition, we introduced next-generation ART using somatic cells with artificial oocytes. Finally, we provided an overview of the reproductive application of in vitro oogenesis and its use in human fertility.

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

Therapeutic Potency of Induced Pluripotent Stem-Cell-Derived Corneal Endothelial-like Cells for Corneal Endothelial Dysfunction.

Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

Efficient hepatic differentiation and regeneration potential under xeno-free conditions using mass-producible amnion-derived mesenchymal stem cells.

BACKGROUND: Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. METHODS: We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. RESULTS: We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. CONCLUSION: This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.

Mitochondrial DNA Haplogroup Related to the Prevalence of Helicobacter pylori.

Mitochondria are essential organelles that are not only responsible for energy production but are also involved in cell metabolism, calcium homeostasis, and apoptosis. Targeting mitochondria is a key strategy for bacteria to subvert host cells' physiology and promote infection. Helicobacter (H.) pylori targets mitochondria directly. However, mitochondrial genome (mtDNA) polymorphism (haplogroup) is not yet considered an important factor for H. pylori infection. Here, we clarified the association of mitochondrial haplogroups with H. pylori prevalence and the ability to perform damage. Seven mtDNA haplogroups were identified among 28 H. pylori-positive subjects. Haplogroup B was present at a higher frequency and haplotype D at a lower one in the H. pylori population than in that of the H. pylori-negative one. The fibroblasts carrying high-frequency haplogroup displayed a higher apoptotic rate and diminished mitochondrial respiration following H. pylori infection. mtDNA mutations were accumulated more in the H. pylori-positive population than in that of the H. pylori-negative one in old age. Among the mutations, 57% were located in RNA genes or nonsynonymous protein-coding regions in the H. pylori-positive population, while 35% were in the H. pylori-negative one. We concluded that gastric disease caused by Helicobacter virulence could be associated with haplogroups and mtDNA mutations.

Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats.

Retinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2-3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.

De Novo Development of mtDNA Deletion Due to Decreased POLG and SSBP1 Expression in Humans.

Defects in the mitochondrial genome (mitochondrial DNA (mtDNA)) are associated with both congenital and acquired disorders in humans. Nuclear-encoded DNA polymerase subunit gamma (POLG) plays an important role in mtDNA replication, and proofreading and mutations in POLG have been linked with increased mtDNA deletions. SSBP1 is also a crucial gene for mtDNA replication. Here, we describe a patient diagnosed with Pearson syndrome with large mtDNA deletions that were not detected in the somatic cells of the mother. Exome sequencing was used to evaluate the nuclear factors associated with the patient and his family, which revealed a paternal POLG mutation (c.868C > T) and a maternal SSBP1 mutation (c.320G > A). The patient showed lower POLG and SSBP1 expression than his healthy brothers and the general population of a similar age. Notably, c.868C in the wild-type allele was highly methylated in the patient compared to the same site in both his healthy brothers. These results suggest that the co- deficient expression of POLG and SSBP1 genes could contribute to the development of mtDNA deletion.

Hepatogenic Potential and Liver Regeneration Effect of Human Liver-derived Mesenchymal-Like Stem Cells.

Human liver-derived stem cells (hLD-SCs) have been proposed as a possible resource for stem cell therapy in patients with irreversible liver diseases. However, it is not known whether liver resident hLD-SCs can differentiate toward a hepatic fate better than mesenchymal stem cells (MSCs) obtained from other origins. In this study, we compared the differentiation ability and regeneration potency of hLD-SCs with those of human umbilical cord matrix-derived stem cells (hUC-MSCs) by inducing hepatic differentiation. Undifferentiated hLD-SCs expressed relatively high levels of endoderm-related markers (GATA4 and FOXA1). During directed hepatic differentiation supported by two small molecules (Fasudil and 5-azacytidine), hLD-SCs presented more advanced mitochondrial respiration compared to hUC-MSCs. Moreover, hLD-SCs featured higher numbers of hepatic progenitor cell markers on day 14 of differentiation (CPM and CD133) and matured into hepatocyte-like cells by day 7 through 21 with increased hepatocyte markers (ALB, HNF4A, and AFP). During in vivo cell transplantation, hLD-SCs migrated into the liver of ischemia-reperfusion injury-induced mice within 2 h and relieved liver injury. In the thioacetamide (TAA)-induced liver injury mouse model, transplanted hLD-SCs trafficked into the liver and spontaneously matured into hepatocyte-like cells within 14 days. These results collectively suggest that hLD-SCs hold greater hepatogenic potential, and hepatic differentiation-induced hLD-SCs may be a promising source of stem cells for liver regeneration.

The Rho-associated kinase inhibitor fasudil can replace Y-27632 for use in human pluripotent stem cell research.

Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders the maintenance and differentiation of stem cells. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative and is cheaper than Y-27632. It increased hPSC growth following thawing and passaging, like Y-27632, and did not affect pluripotency, differentiation ability, and chromosome integrity. Furthermore, fasudil promoted retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells (NCCs) during differentiation. It was also useful for single-cell passaging of hPSCs and during aggregation. These findings suggest that fasudil can replace Y-27632 for use in stem research.