Immune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as “stealth” organoids, hold promise for clinical transplantation. To address immune rejection, we investigated... Show moreImmune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as “stealth” organoids, hold promise for clinical transplantation. To address immune rejection, we investigated the impact of genetically modifying human leukocyte antigen (HLA) class I in kidney organoids prior to transplantation. By using CRISPR-Cas9, we successfully knocked out beta-2-microglobulin (B2M), resulting in iPSCs devoid of HLA class I surface expression. In vitro, the B2M knockout protected kidney organoids derived from these iPSCs against T-cell rejection. To assess in vivo protection, unmodified (control) and B2M–/– kidney organoids were transplanted into humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs). Successful engraftment of human PBMCs was confirmed, and after 4 weeks, we observed no discernible difference in the infiltration rate, proliferation, or cytotoxicity of CD4+ and CD8+ T cells between control and B2M–/– organoids. Both groups of organoids showed compromised tissue integrity, displaying tubulitis and loss of tubule integrity. Notably, while B2M–/– organoids failed to express HLA class I on their cell surface, there was preexisting expression of HLA class II in both control and B2M–/– organoids transplanted into mice with human PBMCs. HLA class II expression was not limited to antigen-presenting cells but also evident in epithelial cells of the kidney organoid, posing an additional immunological challenge to its transplantation. Consequently, we conclude that B2M knockout alone is insufficient to protect iPSC-derived kidney organoids from T-cell-mediated immune rejection. Additionally, our findings suggest that modulating HLA class II signaling will be necessary to prevent rejection following transplantation. Show less
Todtenhaupt, P.; Franken, L.A.; Groene, S.G.; Hoolwerff, M. van; Meeren, L.E. van der; Klink, J.M.M. van; ... ; Pel, M. van 2023
Background aimsHuman umbilical cord–derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods... Show moreBackground aimsHuman umbilical cord–derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, we report on a robust and standardized method for hUC-MSC isolation and expansion.MethodsUsing 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Furthermore, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics.ResultsWe demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of ∼98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity.ConclusionsWe present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies. Show less
Bulut, M.; Cuenca, M.V.; Graaf, M. de; Hil, F.E. van den; Mummery, C.L.; Orlova, V.V. 2022
IntroductionTrophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their... Show moreIntroductionTrophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells. MethodsAfter enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT. ResultsThe trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-alpha and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR. ConclusionWe verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions. Show less
Franceschini, N.; Oosting, J.; Tamsma, M.; Niessen, B.; Briaire-de Bruijn, I.; Akker, B. van den; ... ; Cleton-Jansen, A.M. 2021
For osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are... Show moreFor osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are urgently needed. A hallmark of cancer is aberrant metabolism, which justifies targeting metabolic pathways as a promising therapeutic strategy. One of these metabolic pathways, the NAD+ synthesis pathway, can be considered as a potential target for OS treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the classical salvage pathway for NAD+ synthesis, and NAMPT is overexpressed in OS. In this study, five OS cell lines were treated with the NAMPT inhibitor FK866, which was shown to decrease nuclei count in a 2D in vitro model without inducing caspase-driven apoptosis. The reduction in cell viability by FK866 was confirmed in a 3D model of OS cell lines (n = 3). Interestingly, only OS cells with low nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) RNA expression were sensitive to NAMPT inhibition. Using a publicly available (Therapeutically Applicable Research to Generate Effective Treatments (TARGET)) and a previously published dataset, it was shown that in OS cell lines and primary tumors, low NAPRT1 RNA expression correlated with NAPRT1 methylation around the transcription start site. These results suggest that targeting NAMPT in osteosarcoma could be considered as a novel therapeutic strategy, where low NAPRT expression can serve as a biomarker for the selection of eligible patients. Show less
The second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the... Show moreThe second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the feasibility of using surface markers TNAP, PDPN, EPCAM and ITGA6 to isolate FGCs as well as human primordial germ cell-like cells (hPGCLCs) derived from embryonic stem cells (hESCs) from both sexes by fluorescence-activated cell sorting (FACS). Our results suggest that a combination of TNAP and PDPN was sufficient to separate populations of premeiotic FGCs and hPGCLCs in both sexes. This combination of antibodies also proved efficient in separating 'mitotic' from 'retinoic-acid responsive' female FGCs. Furthermore, we report that the differentiation efficiency of TNAP+PDPN+ hPGCLCs from hESCs was sex-independent, but the ability to propagate differed considerably between the sexes. In contrast to male, female hPGCLCs retained their characteristics and exhibited robust colony-forming ability when cultured for five days in medium containing LIF, forskolin and FGF2. We conclude that marked sex differences exist in the isolation and propagation of human FGCs and hPGCLCs. Our study provides novel insights relevant for the optimization of in vitro gametogenesis in humans. Show less
The adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could replenish functional homeostasis similarly to, e.g., skin or the hematopoietic system. Unlike a mature... Show moreThe adult mammalian kidney is a poorly regenerating organ that lacks the stem cells that could replenish functional homeostasis similarly to, e.g., skin or the hematopoietic system. Unlike a mature kidney, the embryonic kidney hosts at least three types of lineage-specific stem cells that give rise to (a) a ureter and collecting duct system, (b) nephrons, and (c) mesangial cells together with connective tissue of the stroma. Extensive interest has been raised towards these embryonic progenitor cells, which are normally lost before birth in humans but remain part of the undifferentiated nephrogenic rests in the pediatric renal cancer Wilms tumor. Here, we discuss the current understanding of kidney-specific embryonic progenitor regulation in the innate environment of the developing kidney and the types of disruptions in their balanced regulation that lead to the formation of Wilms tumor. Show less
Hsu, C.C.; Xu, J.B.; Brinkhof, B.; Wang, H.; Cui, Z.F.; Huang, W.E.; Ye, H. 2020
Stem cells with the capability to self-renew and differentiate into multiple cell derivatives provide platforms for drug screening and promising treatment options for a wide variety of neural... Show moreStem cells with the capability to self-renew and differentiate into multiple cell derivatives provide platforms for drug screening and promising treatment options for a wide variety of neural diseases. Nevertheless, clinical applications of stem cells have been hindered partly owing to a lack of standardized techniques to characterize cell molecular profiles noninvasively and comprehensively. Here, we demonstrate that a label-free and noninvasive single-cell Raman microspectroscopy (SCRM) platform was able to identify neural cell lineages derived from clinically relevant human induced pluripotent stem cells (hiPSCs). By analyzing the intrinsic biochemical profiles of single cells at a large scale (8,774 Raman spectra in total), iPSCs and iPSC-derived neural cells can be distinguished by their intrinsic phenotypic Raman spectra. We identified a Raman biomarker from glycogen to distinguish iPSCs from their neural derivatives, and the result was verified by the conventional glycogen detection assays. Further analysis with a machine learning classification model, utilizing t-distributed stochastic neighbor embedding (t-SNE)-enhanced ensemble stacking, clearly categorized hiPSCs in different develop- mental stages with 97.5% accuracy. The present study demon-strates the capability of the SCRM-based platform to monitor cell development using high content screening with a noninvasive and label-free approach. This platform as well as our identified bio- marker could be extensible to other cell types and can potentially have a high impact on neural stem cell therapy. Show less
Brink, L. van den; Grandela, C.; Mummery, C.L.; Davis, R.P. 2019
Research on mechanisms underlying monogenic cardiac diseases such as primary arrhythmias and cardiomyopathies has until recently been hampered by inherent limitations of heterologous cell systems,... Show moreResearch on mechanisms underlying monogenic cardiac diseases such as primary arrhythmias and cardiomyopathies has until recently been hampered by inherent limitations of heterologous cell systems, where mutant genes are expressed in noncardiac cells, and physiological differences between humans and experimental animals. Human-induced pluripotent stem cells (hiPSCs) have proven to be a game changer by providing new opportunities for studying the disease in the specific cell type affected, namely the cardiomyocyte. hiPSCs are particularly valuable because not only can they be differentiated into unlimited numbers of these cells, but they also genetically match the individual from whom they were derived. The decade following their discovery showed the potential of hiPSCs for advancing our understanding of cardiovascular diseases, with key pathophysiological features of the patient being reflected in their corresponding hiPSC-derived cardiomyocytes (the past). Now, recent advances in genome editing for repairing or introducing genetic mutations efficiently has enabled the disease etiology and pathogenesis of a particular genotype to be investigated (the present). Finally, we are beginning to witness the promise of hiPSC in personalized therapies for individual patients, as well as their application in identifying genetic variants responsible for or modifying the disease phenotype (the future). In this review, we discuss how hiPSCs could contribute to improving the diagnosis, prognosis, and treatment of an individual with a suspected genetic cardiac disease, thereby developing better risk stratification and clinical management strategies for these potentially lethal but treatable disorders. Show less
To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome... Show moreTo elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in OCT4. Functional manipulation of GATA3 and TFAP2A indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down GATA3 in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis. Show less
The diversity of students’ achievement levels within classrooms has made it essential forteachers to adapt their lessons to the varying educational needs of their students(‘differentiation’).... Show moreThe diversity of students’ achievement levels within classrooms has made it essential forteachers to adapt their lessons to the varying educational needs of their students(‘differentiation’). However, the term differentiation has been interpreted in diverse waysand there is a need to specify what effective differentiation entails. Previous reports oflow to moderate application of differentiation underscore the importance of practicalguidelines for implementing differentiation. In two studies, we investigated how teachersshould differentiate according to experts, as well as the degree to which teachers alreadyapply the recommended strategies. Study 1 employed the Delphi technique and focusgroup discussions to achieve consensus among eleven mathematics experts regarding afeasible model for differentiation in primary mathematics. The experts agreed on a fivestepcycle of differentiation: (1) identification of educational needs, (2) differentiatedgoals, (3) differentiated instruction, (4) differentiated practice, and (5) evaluation ofprogress and process. For each step, strategies were specified. In Study 2, theDifferentiation Self-Assessment Questionnaire (DSAQ) was developed to investigate howteachers self-assess their use of the strategies recommended by the experts. Whileteachers (N = 268) were moderately positive about their application of the strategiesoverall, we also identified areas of relatively low usage (including differentiation forhigh-achieving students) which require attention in teacher professional development.Together, these two studies provide a model and strategies for differentiation in primarymathematics based on expert consensus, the DSAQ which can be employed in futurestudies, and insights into teachers’ self-assessed application of specific aspects ofdifferentiation. Show less
Duggal, G.; Heindryckx, B.; Warrier, S.; Taelman, J.; Jeught, M. van der; Deforce, D.; ... ; Sutter, P. de 2015
In experimental animals and in patients with pulmonary arterial hypertension (PAH), a wide spectrum of structural and functional conditions is known that may be responsible for the switch of a... Show moreIn experimental animals and in patients with pulmonary arterial hypertension (PAH), a wide spectrum of structural and functional conditions is known that may be responsible for the switch of a state of "compensated" right ventricular (RV) hypertrophy to a state of RV failure. In recent years, therapy with differentiated cells, endothelial progenitor cells, and mesenchymal stem cells has been shown to cause partial or complete reversal of pathological characteristics of PAH. The therapeutic effects of stem or progenitor cell therapy are considered to be (1) paracrine effects from stem or progenitor cells that had engrafted in the myocardium (or elsewhere), by compounds that have anti-inflammatory, antiapoptotic, and proangiogenic actions and (2) unloading effects on the right ventricle due to stem or progenitor cell-induced decrease in pulmonary vascular resistance and decrease in pulmonary artery pressure. Show less
Janson, D.; Saintigny, G.; Zeypveld, J.; Mahe, C.; Ghalbzouri, A. el 2014
