Induced Pluripotent Stem Cell-Derived Neural Culture Model of Alzheimer’s Disease: A 3D Organoid Approach

Alzheimer’s disease (AD) is a progressive neurologic disorder that impacts a diverse population of older adults. As three-dimensional (3D) models are powerful tools for advancing AD studies, the authors have been developed AD cortical organoids to enable the observation of AD pathology at the cellular, tissue, and organ levels. For creating the model, APPSwe/Ind (APP) and PSEN1 (PS1) mutant genes were transfected into mouse induced pluripotent stem cells (iPSCs) following which the iPSC lines that expressed mutant APP and PS1 proteins were obtained. Then, using modified serum-free suspended embryoid body culture, AD cerebral organoids were made successfully at various ages. The AD model can show AD’s biochemical and pathological alterations, such as overexpressions of Aβ40 and Aβ42 and a decrease of GABAergic interneurons. The proposed model has the potential for implementation in many biomedical applications, including AD drug screening, stem cell transplant, and neuronal tissue engineering.

Assessment of the In Vitro Cytotoxicity Effects of the Leaf Methanol Extract of Crinum zeylanicum on Mouse Induced Pluripotent Stem Cells and Their Cardiomyocytes Derivatives

Crinum zeylanicum (C. zeylanicum) is commonly used in African folk medicine to treat cardiovascular ailments. In the present study, we investigated the cytotoxic effect of the leaf methanol extract of C. zeylanicum (CZE) using mouse pluripotent stem cells (mPSCs). mPSCs and their cardiomyocytes (CMs) derivatives were exposed to CZE at different concentrations. Cell proliferation, differentiation capacity, and beating activity were assessed using xCELLigence system and microscopy for embryoid body (EB) morphology. Expression of markers associated with major cardiac cell types was examined by immunofluorescence and quantitative RT-PCR. Intracellular reactive oxygen species (ROS) levels were assessed by dichlorodihydrofluorescein diacetate staining. The results showed that the plant extract significantly reduced cell proliferation and viability in a concentration- and time-dependent manner. This was accompanied by a decrease in EB size and an increase in intracellular ROS. High concentrations of CZE decreased the expression of some important cardiac biomarkers. In addition, CZE treatment was associated with poor sarcomere structural organization of CMs and significantly decreased the amplitude and beating rate of CMs, without affecting CMs viability. These results indicate that CZE might be toxic at high concentrations in the embryonic stages of stem cells and could modulate the contracting activity of CMs.

Nox4-IGF2 Axis Promotes Differentiation of Embryoid Body Cells Into Derivatives of the Three Embryonic Germ Layers

Reactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4−/−) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4−/− cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4−/− iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells. Graphical Abstract

Detection of Rh Antibodies in Patient Plasma Using Genome Engineered Induced Pluripotent Stem Cell-Derived Red Cells

Background: Despite transfusion of Rh matched red cells for patients with sickle cell disease, Rh alloimmunization remains a persistent challenge. Rh specificities can be complex, resulting from RH genetic diversity found in patients and donors. Antibody identification is hampered by the lack of appropriate reagent red cells, especially those that can identify antibodies against high prevalence or low prevalence Rh antigens. We used human induced pluripotent stem cells (iPSCs) with the goal of producing renewable red cell reagents to both screen for Rh alloimmunization and to aid complex antibody identification. Methods: We generated a panel of iPSCs that include Rh null, D--, lack the high prevalence antigens hr S or hr B, or express uncommon Rh antigens such as V, VS, Go a, or DAK. For the Rh null line, we used CRISPR/Cas9 genetic engineering to disrupt RHCE via a large deletion in a D- iPSC. For D--, RHD was inserted into the AAVS1 safe harbor locus of an Rh null iPSC line using zinc finger nucleases resulting in a line that constitutively expresses RhD but no RhCE. iPSCs with uncommon variants were reprogrammed from RH genotyped donors or engineered similar to the generation of the D-- line. Hematopoietic differentiation by embryoid body formation was used to generate hematopoietic progenitors that were subsequently cultured towards the erythroid lineage. Mature iRBCs were ficin treated and tested with patient plasma with previously identified Rh antibodies using gel agglutination assays. Results: Rh null iPSC-derived RBCs (iRBCs) showed complete absence of cell surface Rh protein by flow cytometry, while D-- iRBCs showed Rh protein expression levels comparable to D-ce+ iRBCs using an anti-D/CE antibody. We assessed RBC agglutination of Rh null, D--, hr S-, hr B-, VVS+, Go a+, and DAK+ iRBCs using standard Rh typing reagents (Ortho). The reprogrammed uncommon donor iRBCs agglutinated with monoclonal anti-Rh antibodies as predicted by RH genotype, while the Rh null iRBCs showed no agglutination with all 5 common Rh antibodies and D-- iRBCs showed agglutination with anti-D reagents only. Rh null iRBCs showed no agglutination against patient plasma containing anti-D, while D-- iRBCs agglutinated. While D- RHCE*ce homozygous iRBCs showed strong agglutination against patient plasma containing anti-hr S, Rh null, D--, and hr S- iRBCs did not agglutinate. No iRBCs showed agglutination by plasma containing anti-V/VS while VVS+ iRBCs showed strong agglutination. Similarly, no iRBCs showed agglutination by plasma containing anti-Go a while Go a+ iRBCs showed strong agglutination. Detection of most antibodies against Rhce on iRBCs was enhanced by ficin treatment whereas antibodies with D specificity did not require ficin treated cells for detection. Conclusion: We suggest that genetically engineered iPSCs expressing uncommon Rh antigen phenotypes that are difficult or impossible to obtain from red cell donors can expedite antibody identification. Rh null and D-- iRBCs could be useful to discriminate antibodies against RhD versus RhCE. Customized iPSCs that lack high prevalence or express low prevalence Rh antigens could potentially standardize antibody evaluation in patients with complex Rh specificities. Disclosures No relevant conflicts of interest to declare.

Disruption of RING and PHD Domains of TRIM28 Evokes Differentiation in Human iPSCs

TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells’ (ESC) self-renewal potential. As the epigenetic factor modulating chromatin structure, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. Because of many similarities between highly dedifferentiated cancer cells and normal pluripotent stem cells, we applied human induced pluripotent stem cells (hiPSC) as a model for stemness studies. For the first time in hiPSC, we analyzed the function of individual TRIM28 domains. Here we demonstrate the essential role of a really interesting new gene (RING) domain and plant homeodomain (PHD) in regulating pluripotency maintenance and self-renewal capacity of hiPSC. Our data indicate that mutation within the RING or PHD domain leads to the loss of stem cell phenotypes and downregulation of the FGF signaling. Moreover, impairment of RING or PHD domain results in decreased proliferation and impedes embryoid body formation. In opposition to previous data indicating the impact of phosphorylation on TRIM28 function, our data suggest that TRIM28 phosphorylation does not significantly affect the pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with disrupted RING and PHD functions display downregulation of genes associated with tumor metastasis, which are considered important targets in cancer treatment. Our data suggest the potential use of RING and PHD domains of TRIM28 as targets in cancer therapy.

Full methylation of H3K27 by PRC2 is dispensable for initial embryoid body formation but required to maintain differentiated cell identity

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The nature of that requirement is not clear. H3K27me3 is associated with repressed genes, however the modification is not sufficient to induce repression, and in some instances is not required. We blocked full methylation of H3K27 with both a small molecule inhibitor, GSK343 and by introducing a point mutation into EZH2, the catalytic subunit of PRC2. Cells with substantively decreased H3K27 methylation differentiate into embryoid bodies, which contrasts with EZH2 null cells. PRC2 targets had varied requirements for H3K27me3, with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype of blocked H3K27 methylation was an inability of altered cells to maintain a differentiated state when challenged. This phenotype was determined by H3K27 methylation in embryonic stem cells through the first four days of differentiation. Full H3K27 methylation therefore was not necessary for formation of differentiated cell states during embryoid body formation but was required to maintain a stable differentiated state.

Gene Edited Fluorescent Cerebral Organoids to Study Human Brain Function and Disease

Cerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.

Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified N-Acetylmannosamine (ManNAc) Analogs

This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac5ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac5ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac5ManNTProp and Ac5ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac5ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac5ManNTProp and Ac5ManNTBut suppressed adipogenic differentiation, with Ac5ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac5ManNTProp and Ac5ManNTBut) for manipulating human stem cells.