Pluripotent Cell Markers Research Articles

Human induced pluripotent stem cell line (FDHSi005-A) derived from a patient with a deep intronic variant in the GNE gene

GlcNAc2-epimerase myopathy is a rare autosomal recessive myopathy characterized by distal involvement in the lower extremities. Our study reprogrammed human-induced pluripotent stem cells from peripheral blood mononuclear cells of a patient with GNE gene deep intronic variant c.862 + 870C>T and c.478C>T compound heterozygous mutations that co-segregated with the disease. The generated iPSCs express pluripotent cell markers with no mycoplasma contamination. Additionally, these iPSCs demonstrated pluripotency, the capacity to differentiate into the three germ layers, and maintained normal karyotypes. Importantly, we identified that these iPSCs possess the same specific mutations as the patient, making them a robust model for studying GNE myopathy and developing potential therapeutic interventions.

Design of iPSC-based cell model to study the functions of the <i>UBE2A</i> gene

BACKGROUND: The UBE2A protein belongs to the E2 family of ubiquitin-binding enzymes involved in the ubiquitination of substrate proteins. UBE2A mutations lead to congenital X-linked mental retardation syndrome-type Nascimento. How UBE2A participates in the central nervous system development is still unknown. AIM: To establish a cell model based on induced pluripotent stem cells (iPSCs) to study the molecular and cellular functions of UBE2A in neurogenesis. METHODS: Using genomic CRISPR-Cas9 editing and lentiviral transduction, a cell model based on iPSCs from two healthy donors was designed. This cell model includes isogenic iPSCs with knockout and inducible hyperexpression of UBE2A. In addition, iPSCs were obtained by reprogramming peripheral blood mononuclear cells of a patient diagnosed with X-linked mental retardation of Nascimento type, which has a deletion spanning the whole UBE2A locus. RESULTS: The obtained iPSCs demonstrate an ESC-like morphology. They express pluripotent cell markers OCT4, SOX2, SSEA-4, and TRA-1-81 and have normal karyotypes. iPSCs with UBE2A knockout or hyperexpression had significantly increased nuclei size compared with the isogenic control. CONCLUSION: The developed iPSC-based cell model can be used for fundamental studies of the functions of UBE2A in neurogenesis.

CDKN2A promoter methylation enhances self-renewal of glioblastoma stem cells and confers resistance to carmustine

BackgroundGlioblastoma, a highly aggressive form of brain cancer, poses significant challenges due to its resistance to therapy and high recurrence rates. This study aimed to investigate the expression and functional implications of CDKN2A, a key tumor suppressor gene, in glioblastoma cells, building upon the existing background of knowledge in this field.MethodQuantitative reverse transcription PCR (qRT-PCR) analysis was performed to evaluate CDKN2A expression in U87 glioblastoma cells compared to normal human astrocytes (NHA). CDKN2A expression levels were manipulated using small interfering RNA (siRNA) and CDKN2A overexpression vector. Cell viability assays and carmustine sensitivity tests were conducted to assess the impact of CDKN2A modulation on glioblastoma cell viability and drug response. Sphere formation assays and western blot analysis were performed to investigate the role of CDKN2A in glioblastoma stem cell (GSC) self-renewal and pluripotency marker expression. Additionally, methylation-specific PCR (MSP) assays and demethylation treatment were employed to elucidate the mechanism of CDKN2A downregulation in U87 cells.ResultCDKN2A expression was significantly reduced in glioblastoma cells compared to NHA. CDKN2A overexpression resulted in decreased cell viability and enhanced sensitivity to carmustine treatment. CDKN2A inhibition promoted self-renewal capacity and increased pluripotency marker expression in U87 cells. CDKN2A upregulation led to elevated protein levels of p16INK4a, p14ARF, P53, and P21, which are involved in cell cycle regulation. CDKN2A downregulation in U87 cells was associated with high promoter methylation, which was reversed by treatment with a demethylating agent.ConclusionOur findings demonstrate that CDKN2A downregulation in glioblastoma cells is associated with decreased cell viability, enhanced drug resistance, increased self-renewal capacity, and altered expression of pluripotency markers. The observed CDKN2A expression changes are mediated by promoter methylation. These results highlight the potential role of CDKN2A as a therapeutic target and prognostic marker in glioblastoma.

Transgenic Lines of Human Induced Pluripotent Stem Cells ICGi022-A-6 and ICGi022-A-7 with Doxycycline-Inducible Variants of Programmable Nuclease AsCas12a

Genome editing in human pluripotent stem cells using programmable nucleases makes it possible to create models of hereditary pathologies using directed transgenesis, gene knockout, and replacement of individual nucleotides in DNA sequences. Using CRISPR/SpCas9-mediated homologous recombination at the AAVS1 locus, clones of human induced pluripotent stem cells (iPSCs) ICGi022-A (Malakhova et al., 2020) were obtained, which carry transgenes of two variants of the nuclease AsCas12a (also known as AsCpf1), recognizing different PAM consensuses, and the reverse doxycycline transgene-dependent transactivator – M2rtTA. For each AsCas12a variant, the lines ICGi022-A-6 (AsCas12a, PAM 5'-TTTV-3') and ICGi022-A-7 (AsCas12a, PAM 5'-TYCV-3') were obtained. Using Western blot analysis, it was shown that the addition of doxycycline to the culture medium causes activation of the expression of AsCas12a(TTTV) and AsCas12a(TYCV) proteins. The resulting transgenic iPSC clones were subjected to molecular and cytogenetic analysis. Using quantitative PCR and immunocytochemical analysis, it was shown that they have a high level of mRNA expression of gene markers of pluripotent cells, namely OCT4, NANOG and SOX2, as well as specific expression of protein marker OCT4, SOX2, SSEA-4 and TRA-1-60. In addition, using iPSCs spontaneous differentiation into embryoid bodies, it was found that transgenic clones can give derivatives of all three primitive germ layers: ectoderm, mesoderm and endoderm. Cytogenetic analysis showed that transgenic iPSC clones have a normal karyotype, 46,XX.

Enhancing Viability of Human Embryonic Stem Cells during Cryopreservation via RGD-REP-Mediated Activation of FAK/AKT/FoxO3a Signaling Pathway.

Cryopreservation is a crucial method for long-term storage and stable allocation of human pluripotent stem cells (hPSCs), which are increasingly being used in various applications. However, preserving hPSCs in cryogenic conditions is challenging due to reduced recovery rates. To address this issue, the Arginine-Glycine-Aspartate (RGD) motif was incorporated into a recombinant elastin-like peptide (REP). Human embryonic stem cells (hESCs) were treated with REP containing RGD motif (RGD-REP) during suspension and cryopreservation, and the survival rate was analyzed. The underlying mechanisms were also investigated. The addition of RGD-REP to the cryopreservation solution improved cell survival and pluripotency marker expression. The improvement was confirmed to be due to the activation of the FAK-AKT cascade by RGD-REP binding to hESC surface interin protein, and consequent inhibition of FoxO3a. The inactivation of FoxO3a reduced the expression of apoptosis-related genes, such as BIM, leading to increased survival of PSCs in a suspension state. RGD-REP, as a ligand for integrin protein, improves the survival and maintenance of hPSCs during cryopreservation by activating survival signals via the RGD motif. These results have potential implications for improving the efficiency of stem cell usage in both research and therapeutic applications.

Human induced pluripotent stem cells derived from a patient with a mutation of SERPINC1 c.236G>A (p.R79H)

Mutation of SERPINC1 is related to the incidence of Inherited antithrombin (AT) deficiency. In this study, we generated a human induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a patient with a mutation of SERPINC1 c.236G>A (p.R79H). The generated iPSCs express pluripotent cell markers with no mycoplasma contamination. Besides, it has a normal female karyotype and could differentiate into all three germ layers in vitro.

The SW480 cell line, overexpressing PIWIL2 gene, maintains the expression of stemness and proliferation genes in the mice xenografts.

This study aims to confirm previous fundamental in vitro findings about the PIWIL2 gene by investigating the effects of its overexpression on cell cycle, proliferation, apoptosis, and stem cell expression markers in colorectal cancer cells (CRC cells) at in vivo level. PIWIL2 has a critical role in maintaining cellular stemness and proliferation. PIWIL2 is an oncogene whose expression in CRC is associated with the occurrence, metastasis, and poor prognosis. SW480 cells harboring expression vectors with/without PIWIL2 were cultured and inoculated in BALB/c nude mice. Tumor formation and growth were monitored every 3 days. On the 28th day after inoculation, the tumors were harvested for their total RNA extraction, and the expression profiling of the candidate genes was performed by Real-time PCR. Our results for the expression profiling of the xenografted tumors showed a significant increase in the expression of cancer stem cell markers, including CD24, CD133, and pluripotency marker SOX2 in the PIWIL2 over-expressing xenografts, compared to the control cell line. Moreover, PIWIL2 dramatically promoted the anti-apoptotic pathway by inducing STAT3 and BCL2-L1 genes in the PIWIL2 over-expressing xenografts, along with the up-regulation of Cyclin D1 and Ki-67 genes. This research supports our prior in vitro findings, highlighting the critical role that PIWIL2 plays in the development of CRC and its substantial promise as a leading candidate for CRC-targeted therapy.

Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.

Blastomere aggregation using phytohemagglutinin-L improves the establishment efficiency of porcine parthenogenesis-derived embryonic stem-like cell lines.

Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 μg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly (p < 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.

Human-induced pluripotent stem cell line (FDHSi001-A) derived from a patient with a CGG repeat expansion in the 5′UTR of GIPC1

Oculopharyngodistal myopathy (OPDM) is a late-onset degenerative muscle disorder characterized by ptosis and weakening of the facial, pharyngeal, and distal limb muscles. Our study reprogrammed human-induced pluripotent stem cells (iPSC) from the peripheral blood mononuclear cells (PBMCs) of a patient with a CGG repeat expansion in the 5′UTR of GIPC1 gene that co-segregated with the disease. The generated iPSCs express the pluripotent cell markers with no mycoplasma contamination. Besides, it showed normal karyotypes and the capacity to differentiate into three germ layers. We also identified that it had the same specific mutation as the patient did.

Human induced pluripotent stem cells derived from a patient with a mutation of FBN1c.1858C > T (p. Pro620Ser)

Mutation of FBN1 has certain relation with the incidence of cranial cervical artery dissection. Our study reprogrammed human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMC) of a patient with a mutation of FBN1c.1858C > T (p. Pro620Ser). The generated iPSCs express pluripotent cell markers with no mycoplasma contamination. Besides, it has normal karyotype and could differentiate into mesoderm, endoderm and neuronal layers. We also identified it has the same specific mutation with our patient.

The stemness of hepatocytes is maintained by high levels of lipopolysaccharide via YAP1 activation

BackgroundThe liver possesses a powerful regeneration ability, which is correlated with the stemness of hepatocytes in the portal vein (PV). However, the mechanism underlying the maintenance of hepatocyte stemness has not been elucidated. Here, we hypothesized that high levels of lipopolysaccharide from the portal vein might maintain the stemness of hepatocytes in the PV area.MethodsFirst, we examined the location of hepatic stem cells and the concentration of lipopolysaccharide (LPS) in the portal vein and inferior vena cava. Then, we assessed the effect of LPS on stemness maintenance in mice by using antibiotics to eliminate LPS and knocking out the LPS receptor, TLR4. In vitro, the effect of LPS on the stemness of hepatocytes was investigated by colony and sphere formation assays and assessment of pluripotent and stem cell marker expression. Furthermore, we studied the mechanism by which LPS regulates the stemness of hepatocytes. Finally, we ligated the portal vein branch to further verify the effect of LPS.ResultsWe found that a high level of LPS from the portal vein was correlated with the location of hepatic stem cells in the PV area, and elimination of LPS by antibiotics inhibited the expression of the stemness marker. LPS promoted colony and sphere formation and induced the upregulation of pluripotent and stem cell markers in AML12 cells. Furthermore, in the reprogramming medium, LPS facilitated the dedifferentiation of mature hepatocytes into hepatic progenitor-like cells, which exhibited a bipotent differentiation capacity in vivo and in vitro. Mechanistically, LPS bound TLR4 to regulate stemness of hepatocytes via the activation of YAP1 signaling, and blockade of YAP1 abolished the LPS-induced cell stemness and upregulation of pluripotent markers.ConclusionsOur study implies a correlation between LPS/TLR4/YAP1 signaling and cell stemness, and LPS was shown to be involved in stemness maintenance of hepatocytes in the PV area. LPS might be used to induce the dedifferentiation of mature hepatocytes into progenitor-like cells for repair of liver injury.

Transplantation of reprogrammed peripheral blood cells differentiates into retinal ganglion cells in the mouse eye with NMDA-induced injury.

The generation of patient-specific induced pluripotent stem cells (iPSCs) holds significant implications for replacement therapy in treating optic neuropathies such as glaucoma. Stem-cell-based therapy targeted at replacing and replenishing retinal ganglion cells is progressing at a fast pace. However, clinical application necessitates an efficient and robust approach for cell manufacturing. Here, we examine whether the embryo body derived from human peripheral blood-derived iPSC can localize into the host retina and differentiate into retinal ganglion cells after transplantation into a glaucoma injury model. Human peripheral blood T cells were isolated and reprogrammed into an induced pluripotent stem cell (TiPSC) line using Sendai virus transduction carrying transcription factors Sox2, Klf4, c-Myc, and Oct4. TiPSCs were differentiated into RGC using neural basal culture. For in vivo studies, embryo bodies derived from TiPSCs (TiPSC-EB) were injected into the vitreous cavity of N-Methyl-d-aspartic acid (NMDA)-treated mice 2 weeks before sacrifice and retinal dissection. Induced pluripotent stem cells generated from human peripheral blood T cells display stem cell morphology and pluripotency markers. Furthermore, RGC-like cells differentiated from TiPSC exhibit extending axons and RGC marker TUJ1. When transplanted intravitreally into NMDA-treated mice, embryo bodies derived from TiPSC survived, migrated, and incorporated into the retina's GCL layer. In addition, TiPSC-EB transplants were able to differentiate into TUJ1 positive RGC-like cells. Retinal ganglion cells can be differentiated using human peripheral blood cells derived iPSC. Transplantation of embryo body derived from TiPSCs into a glaucoma mouse model could incorporate into host GCL and differentiate into RGC-like cells.

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

ABSTRACTThe blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear β-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.This article has an associated First Person interview with the first author of the paper.

Stage-specific response in early mouse embryos exposed to prednisolone in vitro.

Corticosteroids are increasingly being used during the peri-implantation period to treat women with repeated IVF failure and recurrent miscarriage. However, the direct effects of prednisolone (PRDL), one of the commonly used corticosteroids on early embryo development is not understood. To mimic the possible clinical scenario and to understand the embryonic response to direct PRDL exposure, this pilot study was conducted in a mouse model. Cleavage stage embryos exposed to 3 and 30 µM PRDL in vitro were assessed for peri-implantation developmental potential, genetic integrity, inner cell mass (ICM) proliferation and pluripotency markers in the proliferated ICM cells. Exposure to 30 µM PRDL delayed the embryonic progression beyond compaction (P < 0.05) in comparison to vehicle control and, had reduced total cell number (P < 0.001) than all other groups. In addition, 30 µM PRDL exposure resulted in poor hatching potential (P < 0.05) and increased apoptosis in blastocysts (P < 0.05) compared to 3 µM PRDL. On the other hand, completely formed ICM outgrowths were significantly higher (P < 0.05) in 3 µM PRDL compared to control. However, no significant differences were observed in the expression of pluripotency genes. In conclusion, the trend observed in embryos exposed to PRDL in vitro provides important information concerning the use of this drug when treating patients at the peri-implantation phase of IVF cycles. However, the clinical value of this observation on human embryo development needs further research.

ReN VM spheroids in matrix: A neural progenitor three-dimensional in vitro model reveals DYRK1A inhibitors as potential regulators of radio-sensitivity

IntroductionPre-clinical testing of small molecules for therapeutic development across many pathologies relies on the use of in-vitro and in-vivo models. When designed and implemented well, these models serve to predict the clinical outcome as well as the toxicity of the evaluated therapies. The two-dimensional (2D) reductionist approach where cells are incubated in a mono-layer on hard plastic microtiter plates is relatively inexpensive but not physiologically relevant. In contrast, well developed and applied three dimensional (3D) in vitro models could be employed to bridge the gap between 2D in vitro primary screening and expensive in vivo rodent models by incorporating key features of the tissue microenvironment to explore differentiation, cortical development, cancers and various neuronal dysfunctions. These features include an extracellular matrix, co-culture, tension and perfusion and could replace several hundred rodents in the drug screening validation cascade. MethodsHuman neural progenitor cells from middle brain (ReN VM, Merck Millipore, UK) were expanded as instructed by the supplier (Merck Millipore, UK), and then seeded in 96-well low-attachment plates (Corning, UK) to form multicellular spheroids followed by adding a Matrigel layer to mimic extracellular matrix around neural stem cell niche. ReN VM cells were then differentiated via EGF and bFGF deprivation for 7 days and were imaged at day 7. Radiotherapy was mimicked via gamma-radiation at 2Gy in the absence and presence of selected DYRK1A inhibitors Harmine, INDY and Leucettine 41 (L41). Cell viability was measured by AlamarBlue assay. Immunofluorescence staining was used to assess cell pluripotency marker SOX2 and differentiation marker GFAP. ResultsAfter 7 days of differentiation, neuron early differentiation marker (GFAP, red) started to be expressed among the cells expressing neural stem cell marker SOX2 (green). Radiation treatment caused significant morphology change including the reduced viability of the spheroids. These spheroids also revealed sensitizing potential of DYRK1A inhibitors tested in this study, including Harmine, INDY and L41. Discussion & conclusionsCombined with the benefit of greatly reducing the issues associated with in vivo rodent models, including reducing numbers of animals used in a drug screening cascade, cost, ethics, and potential animal welfare burden, we feel the well-developed and applied 3D neural spheroid model presented in this study will provide a crucial tool to evaluate combinatorial therapies, optimal drug concentrations and treatment dosages.

Integrin-EGFR interaction regulates anoikis resistance in colon cancer cells.

Anoikis resistance is an essential property of cancer cells that allow the extra-cellular matrix-detached cells to survive in a suspended state in body fluid in order to metastasize and invade to distant organs. It is known that integrins play an important role in anoikis resistance, but detailed mechanisms are not well understood. Here we report that highly metastatic colon cancer cells showed a higher degree of anoikis resistance than the normal intestinal epithelial cells. These anoikis-resistant cancer cells express high-levels of integrin-α2, β1, and activated EGFR in the anchorage-independent state than the anchorage-dependent state. In contrast, normal intestinal epithelial cells failed to elevate these proteins. Interestingly, a higher co-association of EGFR with integrin-α2β1/-α5β1 was observed on the surface of anoikis-resistant cells. Thus, in the absence of extra-cellular matrix, integrins in association with EGFR activates downstream effectors ERK and AKT and suppress Caspase-3 activation to induce anoikis resistance as was confirmed from the gene-ablation and pharmacological inhibitor studies. Interestingly, these anoikis-resistant cancer cells express high-level of cancer stem cell signatures (CD24, CD44, CD133, EpCAM) and pluripotent stem cell markers (OCT-4, SOX-2, Nanog) as well as drug-resistant pumps (ABCG2, MDR1, MRP1). Altogether, our findings unravel the interplay between integrin-α2β1/-α5β1 and EGFR in anoikisresistance and suggest that the resistant cells are cancer initiating or cancer stem cells, which may serve as a promising target to combat metastasis of cancer.

Defining Human Pluripotency.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

BackgroundDisease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming.MethodsWe have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined.ResultsWe succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.ConclusionThis robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.

Clinical and molecular markers in retinal detachment-From hyperreflective points to stem cells and inflammation.

PurposeRetinal detachment (RD) is one of the most frequently diagnosed ophthalmologic conditions requiring prompt surgical intervention. Combination of proper surgical technique and new diagnostic markers, both clinical and molecular, can help improve the diagnosis and prognosis of RD treatment.Methods12 patients with rhegmatogenous RD (rRD) were included into the study after obtaining patient consent and Regional Ethical Approval (average age: 58.1 ± 17.4 years). OCT was performed before and after 23G vitrectomy for RD. Pure subretinal fluid (SRF) was collected during surgery and analyzed by protein array profiling on a panel of 105 inflammatory cytokines (Human XL Cytokine Array), while the effect of SRF upon human macrophages-driven phagocytosis of apoptotic retinal pigment epithelial (RPE) cells ex vivo was quantified by flow cytometry. Immunohistochemistry (IHC) of retinectomized tissue due to PVR caused by RD was performed to determine presence of markers for microglial cells (CD34), macrophages and activated microglia (CD68), regulator of the immune response to infection (NFkB), progenitor and stem cell marker (Sox2), pluripotency marker (Oct4) and intermediate filament markers (GFAP and Nestin).ResultsOCT of fresh RD patients contained pre-operatively hyper reflective points (HRPs) at the detached neuroretina border and proximal to the RPE layer—their size and number decreased following successful reattachment surgery. IHC of the retinectomized tissue from detached retina due to severe PVR showed presence of cell conglomerates at the detached neuroretina border which were positive for CD68, NFkB, Sox2 and GFAP, less positive for CD47 and Nestin and negative for Oct4 and CD34. The SRF contained at least 37 cytokines with higher, and 4 cytokine with lower concentration compared to that in vitreous from non-RD pathology; when used as conditional medium to human macrophages ex vivo, the SRF doubled their capacity for engulfing dying RPEs.ConclusionsFresh RD can be hallmarked by presence of HRPs at the detached neuroretina border on OCT; the HRPs decrease in size and number after successful reattachment surgery, and likely resemble the macrophage conglomerates seen by IHC. The neuroretina in RD contains progenitor/stem-like cells and signs of inflammatory reaction, while the SRF contains inflammatory cytokines and other factors which increase the ability of professional phagocytes to engulf dying RPE, or for that matter, other dying cells in the retina.