Human Stem Cell Lines Research Articles

Piezoelectric barium titanate/hydroxyapatite composite coatings on Ti-6Al-4V alloy via electrophoretic deposition

Electrophoretic deposition (EPD) is a widely accepted, cost-effective and simple method to obtain conformal coatings of dense nanoparticles via application of a DC voltage. This paper reports the physical and mechanical properties of nanostructured barium titanate/hydroxyapatite (BT/HA) ceramic composites coated onto Ti-6Al-4V alloy via cathodic EPD in various ratios of 40:60, 50:50 and 60:40 (HB4, HB5 and HB6) respectively. Homogenous BT/HA coatings were obtained at a notably low voltage of 10 V. The crystallinity and phase analysis confirmed the formation of tetragonal phase of BT indicating the piezoelectric property. HB6 exhibits maximum piezoelectric coefficient (d33) of 159 pC/N and Vicker's hardness of 242.31 HV. The cross-sectional electron micrographs show well connected and homogeneous coatings with increasing amounts of BT. Human mesenchymal stem cell lines were utilized in biocompatibility experiments, which revealed that HB composites had greater viability of HW-MSCs cells than pure BT, with HB6 exhibiting a maximum cell viability of more than 90 %.

EEF1B2 regulates the proliferation and apoptosis of human spermatogonial stem cell lines through TAF4B

BackgroundSpermatogonial stem cells (SSCs) are essential for male fertility, maintaining sperm production throughout life. While mouse SSCs have been studied extensively, the mechanisms regulating human SSCs are less understood. ObjectivesTo investigate the role of EEF1B2 in regulating human SSC proliferation and apoptosis. Material and methodsSingle cell RNA sequencing (scRNA-seq) analysis was utilized to investigate the differentially expressed genes of SSC. The distribution of EEF1B2 in the human testis was examined using immunofluorescence and immunohistochemistry techniques. Cell proliferation, DNA replication, and self-renewal were analyzed using CCK8, EdU, Western blot, and flow cytometry. RNA sequencing was employed to analyze the downstream target molecules and signaling pathways of EEF1B2. ResultsIn this study, we analyzed single-cell sequencing data from human testicular samples and identified EEF1B2 as a protein highly expressed in SSCs, with expression decreasing during development. Immunohistochemistry and immunofluorescence confirmed this pattern and showed co-localization with the proliferation marker KI67. Knockdown of EEF1B2 in human SSC lines impaired proliferation and viability, reducing self-renewal proteins like PLZF and CCNE1. RNA sequencing revealed decreased TAF4B following EEF1B2 knockdown, which could be rescued by replenishing TAF4B. Testicular SSCs from non-obstructive azoospermia (NOA) patients also showed reduced EEF1B2. Discussion and conclusionOur findings reveal a novel regulatory mechanism involving EEF1B2 and TAF4B in human SSCs, suggesting EEF1B2 deficiency may contribute to male infertility.

Duloxetine enhances PAX6 expression and suppresses innate immune responses in murine LPS-induced corneal inflammation

Background-aimPAX6 is a key regulator of eye development and epithelial homeostasis in the cornea. When deficient, chronic corneal inflammation, neovascularization and limbal stem cell deficiency can occur. Here we investigated the potential of duloxetine, a generic serotonin reuptake inhibitor that can upregulate PAX6 in vitro, for its in vivo activity in the context of corneal inflammation. MethodsDuloxetine tolerance was tested in a human limbal stem cell line and isogenic CRISPR-knockout PAX6+/− cells. C57BL/6-Wildtype mice were administered duloxetine eye drops at concentrations of 1 μM - 2 mM and tested for toxicity and corneal PAX6 expression. In LPS-induced corneal inflammation in mice, duloxetine's effect on PAX6 expression, corneal opacification and inflammatory responses were evaluated by in vivo corneal imaging, immunostaining, and whole-transcriptome microarray analysis. ResultsNo toxicity was observed in vitro for duloxetine concentrations up to 10μΜ. In vivo, duloxetine drops were well-tolerated up to 50 μM. Duloxetine drops at 10μΜ significantly upregulated PAX6 protein levels in the cornea by 30 % within 2 days. In the LPS model, duloxetine resulted in a sustained 33 % PAX6 protein upregulation in the cornea at 7 days, and in reduced opacity within 2 days, accompanied by a significant dampening of IL-17A signaling, neutrophil degranulation, microglial activation, macrophage markers, and MMP expression, despite non-significant changes in total inflammatory cell infiltration. ConclusionShort-term administration of a repurposed generic drug, duloxetine, upregulates PAX6 protein levels in the cornea of mice and exerts an anti-inflammatory activity by dampening innate immune responses.

Comparison of Natural Killer Cells Differentiated from Various Pluripotent Stem Cells.

Allogeneic natural killer (NK) cell therapy has been effective in treating cancer. Many studies have tested NK cell therapy using human pluripotent stem cells (hPSCs). However, the impacts of the origin of PSC-NK cells on competence are unclear. In this study, several types of hPSCs, including human-induced PSCs (hiPSCs) generated from CD34+, CD3-CD56+, and CD56- cells in umbilical cord blood (UCB), three lines of human embryonic stem cells (hESCs, ES-1. ES-2 and ES-3) and MHC I knockout (B2M-KO)-ESCs were used to differentiate into NK cells and their capacities were analyzed. All PSC types could differentiate into NK cells. Among the iPSC-derived NK cells (iPSC-NKs) and ESC-derived NK cells (ES-NKs), 34+ iPSCs and ES-3 had a higher growth rate and cytotoxicity, respectively, ES-3 also showed better efficacy than 34+ iPSCs. B2M-KO was similar to the wild type. These results suggest that the screening for differentiation of PSCs into NK cells prior to selecting the PSC lines for the development of NK cell immunotherapy is an essential process for universal allotransplantation, including the chimeric antigen receptor (CAR).

IS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection.

Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as "triggers" and "targets" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNaseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic "trigger" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous "target" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This lrRNA-seq-informed Tomahto targeted approach is a new modality for generating protein-level evidence of alternative isoforms─a critical first step in designing functional studies and eventually clinical assays.

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Anaesthetics reduce the viability of adipose-derived stem cells

ABSTRACT Adipose-derived stem cells (ADSCs) are characterized by their low immunogenicity and unique immunosuppressive properties, providing many opportunities for autologous transplantation in regenerative medicine and plastic surgery. These methods are characterized by low rejection rates and intense stimulation of tissue regeneration. However, procedures during which fat tissue is harvested occur under local anaesthesia. To better understand the effects and mechanisms of anaesthetic compounds in cosmetic and therapeutic procedures, the present study used a mixture of these compounds (0.1% epinephrine, 8.4% sodium bicarbonate, and 4% articaine) and examined their impact on a human adipose-derived stem cell line. The results showed anesthetics’ negative, dose-dependent effect on cell viability and proliferation, especially during the first 24 h of incubation. After extending the exposure to 48 and 72 h of incubation, cells adapted to new culture conditions. In contrast, no significant changes were observed in immunophenotype, cell cycle progression, and apoptosis. The results obtained from this study provide information on the effect of the selected mixture of anaesthetics on the characteristics and function of ASC52telo cells. The undesirable changes in the metabolic activity of cells suggest the need to search for new drugs to harvest cells with unaltered properties and higher efficacy in aesthetic medicine treatments.

Discovery of YS-1 as a cell line of gastric inflammatory cancer-associated fibroblasts.

Inflammatory cancer-associated fibroblasts (iCAFs) was first identified by co-culture of pancreatic stellate cells and tumor organoids. The key feature of iCAFs is IL-6high/αSMAlow. We examine this phenomenon in gastric cancer using two cell lines of gastric fibroblasts (HGF and YS-1). HGF or YS-1 were co-cultured with MKN7 (a gastric adenocarcinoma cell line) in Matrigel. IL-6 protein levels in the culture supernatant were measured by ELISA. The increased production of IL-6 was not observed in any of the combinations. Instead, the supernatant of YS-1 exhibited the higher levels of IL-6. YS-1 showed IL-6high/αSMA (ACTA2)low in real-time PCR, mRNA-seq and immunohistochemistry. In mRNA-seq, iCAFs-associated genes and signaling pathways were up-regulated in YS-1. No transition to myofibroblastic phenotype was observed by monolayer culture, or the exposure to sonic hedgehog (SHH) or TGF-β. YS-1 conditioned medium induced changes of morphology and stem-ness/differentiation in NUGC-3 (a human gastric adenocarcinoma cell line) and UBE6T-15 (a human bone marrow-derived mesenchymal stem cell line). YS-1 is a stable cell line of gastric iCAFs. This discovery will promote further research on iCAFs for many researchers.

The sirtuin-associated human senescence program converges on the activation of placenta-specific gene PAPPA

Sirtuins are pro-longevity genes with chromatin modulation potential, but how these properties are connected is not well understood. Here, we generated a panel of isogeneic human stem cell lines with SIRT1-SIRT7 knockouts and found that any sirtuin deficiency leads to accelerated cellular senescence. Through large-scale epigenomic analyses, we show how sirtuin deficiency alters genome organization and that genomic regions sensitive to sirtuin deficiency are preferentially enriched in active enhancers, thereby promoting interactions within topologically associated domains and the formation of de novo enhancer-promoter loops. In all sirtuin-deficient human stem cell lines, we found that chromatin contacts are rewired to promote aberrant activation of the placenta-specific gene PAPPA, which controls the pro-senescence effects associated with sirtuin deficiency and serves as a potential aging biomarker. Based on our survey of the 3D chromatin architecture, we established connections between sirtuins and potential target genes, thereby informing the development of strategies for aging interventions.

Dynein and dynactin move long-range but are delivered separately to the axon tip.

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines aremodified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assayprovides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

Microarray evidence that 8-cell human embryos express some hormone family members including oxytocin.

This study is to discover hormone pathways active in early cleaving human embryos. A list of 152 hormones and receptors were compiled to query the microarray database of mRNAs in 8-cell human embryos, two lines of human embryonic stem cells plus human fibroblasts before and after induced pluripotency. Over half of the 152 hormones and receptors were silent on the arrays of all cell types, and more were detected at high or moderate levels on the 8-cell arrays than on the pluripotent cell or fibroblast arrays. Eight hormone family genes were uniquely detected at least 22-fold higher on the 8-cell arrays than the stem cell arrays: AVPI1, CCK, CORT, FSTL4, GIP, GPHA2, OXT, and PPY suggesting novel roles for these proteins in early development. Oxytocin was detected by pilot immunoassay in culture media collected from Day 3 embryos. Robust detection of CRHR1 and EPOR suggests the 8-cell embryo may be responsive to maternal CRH and EPO. The over-expression of POMC and GHITM suggests POMP peptide products may have undiscovered roles in early development and GHITM may contribute to mitochondrial remodeling. Under-detected on the 8-cell arrays at least tenfold were two key enzymes in steroid biosynthesis, DHCR24 and FDPS. The 8-cell human embryo may be secreting oxytocin, which could stimulate its own progress down the fallopian tube as well as play a role in early neural precursor development. The 8-cell embryo does not synthesize reproductive steroid hormones. As previously reported for growth factor families, the early embryo over-expresses more hormones than hormone receptors.

Gα12 signaling regulates transcriptional and phenotypic responses that promote glioblastoma tumor invasion

In silico interrogation of glioblastoma (GBM) in The Cancer Genome Atlas (TCGA) revealed upregulation of GNA12 (Gα12), encoding the alpha subunit of the heterotrimeric G-protein G12, concomitant with overexpression of multiple G-protein coupled receptors (GPCRs) that signal through Gα12. Glioma stem cell lines from patient-derived xenografts also showed elevated levels of Gα12. Knockdown (KD) of Gα12 was carried out in two different human GBM stem cell (GSC) lines. Tumors generated in vivo by orthotopic injection of Gα12KD GSC cells showed reduced invasiveness, without apparent changes in tumor size or survival relative to control GSC tumor-bearing mice. Transcriptional profiling of GSC-23 cell tumors revealed significant differences between WT and Gα12KD tumors including reduced expression of genes associated with the extracellular matrix, as well as decreased expression of stem cell genes and increased expression of several proneural genes. Thrombospondin-1 (THBS1), one of the genes most repressed by Gα12 knockdown, was shown to be required for Gα12-mediated cell migration in vitro and for in vivo tumor invasion. Chemogenetic activation of GSC-23 cells harboring a Gα12-coupled DREADD also increased THBS1 expression and in vitro invasion. Collectively, our findings implicate Gα12 signaling in regulation of transcriptional reprogramming that promotes invasiveness, highlighting this as a potential signaling node for therapeutic intervention.

EXTH-65. IDENTIFICATION OF LOMERIZINE AS A POTENTIAL ANTI-GLIOBLASTOMA DRUG

Abstract BACKGROUND Glioblastoma (GBM) is one of the most malignant primary brain tumors. Despite development of surgical techniques, chemotherapy and radiotherapy, GBM prognosis is still poor. Here, we identified lomerizine, a prophylactic drug for migraine, as a potential anti-GBM drug from thousands of existing compounds. We investigated its therapeutic effects both in vitro and in vivo with the purpose of drug repositioning. Materials and method: Human patient-derived glioma stem cell line (GSC; KGS01, KGS10, KGS15), their differentiated cells, and GBM cell lines (A172, SNB19, T98, U87) were used. Proliferation assay, migration assay and invasion assay were performed with all cell lines. Sphere-forming assay was performed with GSCs. Various signaling pathways altered by lomerizine were analyzed by Western blotting. In addition, induction of apoptosis was evaluated by immunofluorescence and Annexin V assay. Anti-GBM effects of lomerizine in vivo was validated with a mouse brain tumor model. Result: Lomerizine inhibited proliferation, migration, and invasion dose-dependently in all cell lines, especially in GSCs. Sphere formation was also suppressed by lomerizine in all GSCs. STAT3, which is involved in tumorigenesis, was dephosphorylated after the treatment with lomerizine in a dose-dependent manner in all cell lines. Furthermore, apoptosis induction by lomerizine was confirmed in both immunofluorescence and Annexin V assay. In vivo experiments showed a significant tumor suppression and prolongation of overall survival in the group of mice treated with lomerizine. CONCLUSION This study suggests that lomerizine has anti- GBM effects.

OR14-02 The Thyroid Hormone Analogs DITPA And TRIAC Triggered T3-signalling In Human MCT8-deficient Brain Organoids

Abstract Disclosure: F. Salas Lucia: None. A.M. Dumitrescu: None. S. Refetoff: None. Patients with mutations in the X chromosome-linked thyroid hormone (TH) cell transporter MCT8 gene have brain hypothyroidism, resulting in severe neuro-psychomotor retardation for which treatment remains very challenging. Potential new treatments using the TH-analogues 3,5-diiodothyropropionic acid (DITPA) and 3,3’,5-triiodothyroacetic acid (TRIAC) have been explored in Mct8-deficient mice. However, given species differences in brain TH transporters, in vitro models using human cells are needed. Here, we generated human brain organoids (BOs) from 4 lines of human induced pluripotent stem cells (hiPSCs). Two lines were derived from MCT8-deficient patients. The other two lines were used as controls: one isogenic line in which the MCT8 mutation has been corrected using CRISPR/Cas9, and a line derived from the unaffected father. Control BOs showed multiple large and continuous neuroepithelia surrounding a ventricle and expressed forebrain markers (FOXG1, and LHX2) at 10-fold higher levels than the hiPSCs. After 20 days in culture, BOs expressed TH transporters; the most abundant being MCT8, trailed by LAT2, LAT1, and MCT10. In contrast, the MCT8-deficient BOs exhibit a 3-fold decrease in the relative expression of LAT2, compared to controls. All BOs showed a similar expression of the deiodinase type 3 (DIO3), and the TH nuclear receptor beta (THRB) genes; the DIO2 and THRA expression were undetectable. Additionally, all four BOs expressed the neuronal and glial markers NEUN and GFAP (5.1±2.0, and 7.3±3.8-fold expression, respectively). Immunohistochemistry using fluorescent antibodies confirmed the presence of postmitotic neurons (TUJ1+) expressing MCT8 in the BOs. Next, we measured the D3 activity. BOs were incubated with T3I125, which resulted in a prominent peak of T2I125 measured using an ultra-high-performance liquid chromatography linked to a gamma counter. The peak of T2I125 indicates the uptake of T3I125 into the neural cells residing in the BOs, its metabolism, and the release of T2I125 in the medium. MCT8-deficient BOs exhibited a D3 activity of 5.3±3.1 pmol/mg/h, a 70% less when compared to the 18.4±7.7 pmol/mg/h of controls. When control BOs were incubated with the MCT8 inhibitor Silychristin 2µM, they exhibited 4.4±3.0 pmol/mg/h D3 activity, like MCT8-deficient BOs. Next, BOs were incubated for 24h with either 10nM L-T3, 10nM TRIAC, or 3.5μM DITPA and T3-responsive genes were studied. Treatment with L-T3 upregulated by 2-fold the gene KLF9 in all four BOs, suggesting that the T3-signalling in the MCT8-deficient BOs might not be compromised. The genes HAIRLESS and DIO3 were unresponsive. In parallel, incubation with DITPA and TRIAC also upregulated KLF9 by 1.5-fold, representing a proof of concept for these thyromimetic molecules to act in human neural cells. This human cell model may be used as a platform to screen therapeutic drugs for MCT8 deficiency. Presentation: Friday, June 16, 2023

FRI604 Stem-cell Derived Islets For Treating Diabetes

Abstract Disclosure: W.L. Rust: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. P.M. Dalal: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. A.P. Koval: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. J.J. Ratiu: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. S.M. Southard: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. P.S. Strumph: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. C.A. Welsch: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. Background and Aims: We report the development of a novel human multipotent immortal stem cell line, SR1423 that efficiently differentiates to clusters of functional endocrine pancreatic cells. These Synthetic Replacement Endocrine (SRE) clusters contain cell types that recapitulate native islet function in vitro and potently control blood glucose in animal models of type 1 diabetes. Detailed characterization reveals significant distinctions between SRE, native islets and SC-beta cells. SRE clusters are manufactured following GMP requirements in closed vessels with large-scale formats and are being developed for near-term clinical studies. Materials and Methods: SRE was generated by transient expression of the Yamanaka factors in human islet cells harvested from a consented donor pancreas. Rather than screen for pluripotency, this cell line was selected for its ability to differentiate to the definitive endoderm in a first screen, and Pdx1+ pancreatic progenitors in a second screen. SRE does not meet criteria for pluripotency as it fails to express the master control gene for mesoderm specification, brachyury, in response to mesoderm-inducing agents. Comparison against a pluripotent stem cell database demonstrates poor overall differentiation capability for the mesoderm lineage. In contrast, SRE responds to basic differentiation protocols that drive endocrine pancreatic fate choice and generates highly pure populations of cells with endocrine cell characteristics. We therefore characterize SR1423 as a multipotent, and not pluripotent stem cell. In a prospectively designed islet replacement study male NSG mice with streptozotocin-induced diabetes were implanted with SRE clusters to the kidney capsule or gonadal fat pad (as a surrogate for the omentum) and disease progression was monitored. Results: SRE resemble human islets in morphology and distribution of hormone expressing cells. In this mouse model of STZ-induced type 1 diabetes (blood glucose > 180 mg/dL), SRE transplantation resulted in euglycemia 3-4 weeks post-implant; 72 mg/dL +/− 12 SEM when implanted to the kidney capsule (n=13), and 79 mg/dL +/− 15 SEM when implanted to the gonadal fat pad (n=10). Control mice (without SRE implant) in this model of type 1 diabetes (n=2) remained hyperglycemic; 570 mg/dL +/− 20 SEM. Conclusion: These data are consistent with demonstration of proof of principle in this model with resolution of hyperglycemia after transplant of SRE. This is the first description of a novel cell type (non-native, non-SC-beta) with potential clinical therapeutic benefit. SRE clusters are currently in preparation for clinical studies. Presentation: Friday, June 16, 2023

Generation of two iPSC lines from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12

We generated two human induced pluripotency stem cell (hiPSC) lines, RCMGi011-A and 11-B, from skin fibroblast from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12 using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit. We verified variant c.808 T > G and insertion in GLB1 gene, as well as two mutations, c.6992 T > C and c.805C > T, in CDH23 gene which lead to autosomal recessive hearing loss type 12. We have demonstrated normal karyotype of hiPSCs and capacity for cell differentiation into three germ layers.

Assessing non-synthetic crosslinkers in biomaterial inks based on polymers of marine origin to increase the shape fidelity in 3D extrusion printing

In the past decade, there has been significant progress in 3D printing research for tissue engineering (TE) using biomaterial inks made from natural and synthetic compounds. These constructs can aid in the regeneration process after tissue loss or injury, but achieving high shape fidelity is a challenge as it affects the construct’s physical and biological performance with cells. In parallel with the growth of 3D bioprinting approaches, some marine-origin polymers have been studied due to their biocompatibility, biodegradability, low immunogenicity, and similarities to human extracellular matrix components, making them an excellent alternative to land mammal-origin polymers with reduced disease transmission risk and ethical concerns. In this research, collagen from shark skin, chitosan from squid pens, and fucoidan from brown algae were effectively blended for the manufacturing of an adequate biomaterial ink to achieve a printable, reproducible material with a high shape fidelity and reticulated using four different approaches (phosphate-buffered saline, cell culture medium, 6% CaCl2, and 5 mM Genipin). Materials characterization was composed by filament collapse, fusion behavior, swelling behavior, and rheological and compressive tests, which demonstrated favorable shape fidelity resulting in a stable structure without deformations, and interesting shear recovery properties around the 80% mark. Additionally, live/dead assays were conducted in order to assess the cell viability of an immortalized human mesenchymal stem cell line, seeded directly on the 3D printed constructs, which showed over 90% viable cells. Overall, the Roswell Park Memorial Institute cell culture medium promoted the adequate crosslinking of this biopolymer blend to serve the TE approach, taking advantage of its capacity to hamper pH decrease coming from the acidic biomaterial ink. While the crosslinking occurs, the pH can be easily monitored by the presence of the indicator phenol red in the cell culture medium, which reduces costs and time.

Hierarchically Porous Implants Orchestrating a Physiological Viscoelastic and Piezoelectric Microenvironment for Bone Regeneration.

The extracellular matrix microenvironment of bone tissue comprises several physiological cues. Thus, artificial bone substitute materials with a single cue are insufficient to meet the demands for bone defect repair. Regeneration of critical-size bone defects remains challenging in orthopedic surgery. Intrinsic viscoelastic and piezoelectric cues from collagen fibers play crucial roles in accelerating bone regeneration, but scaffolds or implants providing integrated cues have seldom been reported. In this study, we aimed to design and prepare hierarchically porous poly(methylmethacrylate)/polyethyleneimine/poly(vinylidenefluoride) composite implants presenting a similar viscoelastic and piezoelectric microenvironment to bone tissue via anti-solvent vapor-induced phase separation. The viscoelastic and piezoelectric cues of the composite implants for human bone marrow mesenchymal stem cell line stimulated and activated Piezo1 proteins associated with mechanotransduction signaling pathways. Cortical and spongy bone exhibited excellent regeneration and integration in models of critical-size bone defects on the knee joint and femur in vivo. This study demonstrated that implants with integrated physiological cues were promising artificial bone substitute materials for regenerating critical-size bone defects. This article is protected by copyright. All rights reserved.