Weeks Of Differentiation Research Articles

Human-Induced Pluripotent Stem Cell (iPSC)-Derived GABAergic Neuron Differentiation in Bipolar Disorder.

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis.

Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs). Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone). Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment. Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.

Morphological diversification and functional maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain.

Astrocytes, the most abundant glial cell type in the brain, are underrepresented in traditional cortical organoid models due to the delayed onset of cortical gliogenesis. Here we introduce a new glia-enriched cortical organoid model that exhibits accelerated astrogliogenesis. We demonstrated that induction of a gliogenic switch in a subset of progenitors enabled the rapid derivation of astroglial cells, which account for 25-31% of the cell population within 8-10 weeks of differentiation. Intracerebral transplantation of these organoids reliably generated a diverse repertoire of cortical neurons and anatomical subclasses of human astrocytes. Spatial transcriptome profiling identified layer-specific expression patterns among distinct subclasses of astrocytes within organoid transplants. Using an in vivo acute neuroinflammation model, we identified a subpopulation of astrocytes that rapidly activates pro-inflammatory pathways upon cytokine stimulation. Additionally, we demonstrated that CD38 signaling has a crucial role in mediating metabolic and mitochondrial stress in reactive astrocytes. This model provides a robust platform for investigating human astrocyte function.

Distinctive In Vitro Phenotypes in iPSC-Derived Neurons From Patients With Gain- and Loss-of-Function SCN2A Developmental and Epileptic Encephalopathy.

SCN2A encodes NaV1.2, an excitatory neuron voltage-gated sodium channel and a major monogenic cause of neurodevelopmental disorders, including developmental and epileptic encephalopathies (DEE) and autism. Clinical presentation and pharmocosensitivity vary with the nature of SCN2A variant dysfunction and can be divided into gain-of-function (GoF) cases with pre- or peri-natal seizures and loss-of-function (LoF) patients typically having infantile spasms after 6 months of age. We established and assessed patient induced pluripotent stem cell (iPSC) - derived neuronal models for two recurrent SCN2A DEE variants with GoF R1882Q and LoF R853Q associated with early- and late-onset DEE, respectively. Two male patient-derived iPSC isogenic pairs were differentiated using Neurogenin-2 overexpression yielding populations of cortical-like glutamatergic neurons. Functional properties were assessed using patch clamp and multielectrode array recordings and transcriptomic profiles obtained with total mRNA sequencing after 2-4 weeks in culture. At 3 weeks of differentiation, increased neuronal activity at cellular and network levels was observed for R1882Q iPSC-derived neurons. In contrast, R853Q neurons showed only subtle changes in excitability after 4 weeks and an overall reduced network activity after 7 weeks in vitro. Consistent with the reported efficacy in some GoF SCN2A patients, phenytoin (sodium channel blocker) reduced the excitability of neurons to the control levels in R1882Q neuronal cultures. Transcriptomic alterations in neurons were detected for each variant and convergent pathways suggested potential shared mechanisms underlying SCN2A DEE. In summary, patient iPSC-derived neuronal models of SCN2A GoF and LoF pathogenic variants causing DEE show specific functional and transcriptomic in vitro phenotypes.

Degree of differentiation impacts neurobiological signature and resistance to hypoxia of SH-SY5Y cells

Objective. SH-SY5Y cells are valuable neuronal in vitro models for studying patho-mechanisms and treatment targets in brain disorders due to their easy maintenance, rapid expansion, and low costs. However, the use of various degrees of differentiation hampers appreciation of results and may limit the translation of findings to neurons or the brain. Here, we studied the neurobiological signatures of SH-SY5Y cells in terms of morphology, expression of neuronal markers, and functionality at various degrees of differentiation, as well as their resistance to hypoxia. We compared these to neurons derived from human induced pluripotent stem cells (hiPSCs), a well-characterized neuronal in vitro model. Approach. We cultured SH-SY5Y cells and neurons derived from hiPSCs on glass coverslips or micro-electrode arrays. We studied expression of mature neuronal markers, electrophysiological activity, and sensitivity to hypoxia at various degrees of differentiation (one day up to three weeks) in SH-SY5Y cells. We used hiPSC derived neurons as a reference. Main results. Undifferentiated and shortly differentiated SH-SY5Y cells lacked neuronal characteristics. Expression of neuronal markers and formation of synaptic puncta increased during differentiation. Longer differentiation was associated with lower resistance to hypoxia. At three weeks of differentiation, MAP2 expression and vulnerability to hypoxia were similar to hiPSC-derived neurons, while the number of synaptic puncta and detected events were significantly lower. Our results show that at least three weeks of differentiation are necessary to obtain neurobiological signatures that are comparable to those of hiPSC-derived neurons, as well as similar sensitivities to metabolic stress. Significance. This indicates that extended differentiation protocols should be used to study neuronal characteristics and to model brain disorders with SH-SY5Y cells. We provided insights that may offer the basis for the utilization of SH-SY5Y cells as a more relevant neuronal model in the study of brain disorders.

Development of an accelerated cellular model for early changes in Alzheimer’s disease

Alzheimer’s Disease (AD) is a leading cause of dementia characterized by amyloid plaques and neurofibrillary tangles, and its pathogenesis remains unclear. Current cellular models for AD often require several months to exhibit phenotypic features due to the lack of an aging environment in vitro. Lamin A is a key component of the nuclear lamina. Progerin, a truncated protein resulting from specific lamin A mutations, causes Hutchinson–Gilford Progeria Syndrome (HGPS), a disease that prematurely ages individuals. Studies have reported that lamin A expression is induced in the brains of AD patients, and overlapping cellular phenotypes have been observed between HGPS and AD cells. In this study, we investigated the effects of exogenous progerin expression on neural progenitor cells carrying familial AD mutations (FAD). Within three to four weeks of differentiation, these cells exhibited robust AD phenotypes, including increased tau phosphorylation, amyloid plaque accumulation, and an elevated Aβ42 to Aβ40 ratio. Additionally, progerin expression significantly increased AD cellular phenotypes such as cell death and cell cycle re-entry. Our results suggest that progerin expression could be used to create an accelerated model for AD development and drug screening.

Identification of TEKTIN1-expressing multiciliated cells during spontaneous differentiation of non-human primate embryonic stem cells.

Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.

The expression of anti-aging protein Klotho is increased during neural differentiation of bone marrow-derived mesenchymal stem cells.

Klotho, as an antiaging protein, is involved in the maintenance and differentiation of neuronal or glial cells and, therefore, has been noticed as a potential therapeutic target for neurodegenerative disorders. Expression of Klotho has been examined in different cells and organs, however, our information about the developmental pattern of this protein during differentiation of mesenchymal stem cells (MSCs) into neuron-like cells is limited. In this study, we conducted neural differentiation of mouse bone marrow-derived-MSCs and monitored the expression of Klotho together with selected neuron-specific genes at messenger RNA (mRNA) on days 7 and 14 of differentiation using quantitative real-time PCR. In addition, Klotho status at protein level was evaluated by immunocytochemistry. The results showed a significant change in the morphology of MSCs towards neuron-like cells. These changes were observed with progressive growth and formation of cell connections towards the formation of a chain of neuron-like cells which occurred in the second week of differentiation. Morphological changes were associated with a significant increase in the expression of neuron-specific genes like pax-6, neuN and, neurofilaments (NfL). Likewise, there was an increased expression of Klotho mRNA, and accumulation of Klotho protein in neuronal cell bodies, during the cellular differentiation of MSCs. These findings provided new evidence that neuronal differentiation from the MSCs is associated with increased expression of Klotho. These data may provide insight into the importance of Klotho protein in stem cell differentiation and regeneration in response to cell death in the central nervous system.

Incorporating microglia-like cells in human induced pluripotent stem cell-derived retinal organoids.

Microglia are the primary resident immune cells in the retina. They regulate neuronal survival and synaptic pruning making them essential for normal development. Following injury, they mediate adaptive responses and under pathological conditions they can trigger neurodegeneration exacerbating the effect of a disease. Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are increasingly being used for a range of applications, including disease modelling, development of new therapies and in the study of retinogenesis. Despite many similarities to the retinas developed in vivo, they lack some key physiological features, including immune cells. We engineered an hiPSC co-culture system containing retinal organoids and microglia-like (iMG) cells and tested their retinal invasion capacity and function. We incorporated iMG into retinal organoids at 13 weeks and tested their effect on function and development at 15 and 22 weeks of differentiation. Our key findings showed that iMG cells were able to respond to endotoxin challenge in monocultures and when co-cultured with the organoids. We show that retinal organoids developed normally and retained their ability to generate spiking activity in response to light. Thus, this new co-culture immunocompetent in vitro retinal model provides a platform with greater relevance to the in vivo human retina.

Development of lacrimal gland organoids from iPSC derived multizonal ocular cells.

Lacrimal gland plays a vital role in maintaining the health and function of the ocular surface. Dysfunction of the gland leads to disruption of ocular surface homeostasis and can lead to severe outcomes. Approaches evolving through regenerative medicine have recently gained importance to restore the function of the gland. Using human induced pluripotent stem cells (iPSCs), we generated functional in vitro lacrimal gland organoids by adopting the multi zonal ocular differentiation approach. We differentiated human iPSCs and confirmed commitment to neuro ectodermal lineage. Then we identified emergence of mesenchymal and epithelial lacrimal gland progenitor cells by the third week of differentiation. Differentiated progenitors underwent branching morphogenesis in the following weeks, typical of lacrimal gland development. We were able to confirm the presence of lacrimal gland specific acinar, ductal, and myoepithelial cells and structures during weeks 4-7. Further on, we demonstrated the role of miR-205 in regulation of the lacrimal gland organoid development by monitoring miR-205 and FGF10 mRNA levels throughout the differentiation process. In addition, we assessed the functionality of the organoids using the β-Hexosaminidase assay, confirming the secretory function of lacrimal organoids. Finally, metabolomics analysis revealed a shift from amino acid metabolism to lipid metabolism in differentiated organoids. These functional, tear proteins secreting human lacrimal gland organoids harbor a great potential for the improvement of existing treatment options of lacrimal gland dysfunction and can serve as a platform to study human lacrimal gland development and morphogenesis.

Characterizing a novel retinal organoid model of Oculocutaneous albinism and optic nerve misrouting

Albinism refers to a group of genetic disorders typically characterized by a loss/reduction of melanin in the hair, skin and eyes of affected patients. Apart from pigment changes, all albinism patients present with foveal hypoplasia and optic nerve misrouting, and have blurred vision. The molecular mechanisms that link this lack of pigment with neural retinal development are poorly understood, with foveal and optic tract development being difficult to model. To advance our knowledge, we developed a novel retinal organoid model of albinism, and characterized the development and outgrowth of retinal ganglion cells affected during albinism as a model for future studies. Human oculocutaneous albinism 1 (OCA1) patient-derived stem cells were differentiated alongside controls into retinal organoids, as published previously1,2 . Early retinal ganglion cells develop in the first 4 weeks of differentiation, at which point whole organoids could be plated to allow for optic nerve-like outgrowth. Whole organoids were also fixed and analysed with immunohistochemistry (IHC) to visualize contralateral and ipsilateral ganglion cells present in the organoid. IHC analysis showed differences in the number of ipsilateral and contralateral retinal ganglion cells between the healthy control and albinism organoids, in line with in vivo observations. Further, optic nerve-like outgrowth could be achieved with both models, allowing for future research into optic nerve misrouting in albinism. We generated a novel retinal organoid model of oculocutaneous albinism, and characterized the retinal ganglion cell development and outgrowth. This will allow us in the future to study a different and sometimes overlooked aspect of albinism; optic nerve misrouting.

Silk Bone Marrow-like Niches for Red Cell Production Ex Vivo

Background Human adults produce up to 3 million red blood cells every second, equating to about 200 billion generated every day (Caulier and Sankaran Blood 2022). Leading studies point to the erythroblastic islands as the core of red blood cell production within the bone marrow microenvironment. Here, cellular, chemical, and physical elements define a specialized three-dimensional (3D) niche that orchestrates lineage commitment, cell maturation, and final enucleation. The extracorporeal generation of physiologically relevant models of the bone marrow is central to multiple healthcare challenges including studying the physio-pathological mechanisms of human erythropoiesis, personalized drug screening, and production of red blood cells on-demand. Different culture protocols have been developed, aiming at the mass production of erythrocytes in vitro, including bioengineering techniques for developing physiologically relevant models of the hematopoietic niche (Di Buduo et al. Haematologica 2021). Extrapolating the 3D structure and function of the erythroblastic islands is still challenging. Consequently, enucleation remains the main critical limiting step of efficient erythrocyte production ex vivo. Aim We have pioneered bone marrow tissue engineering by using silk fibroin, derived from Bombyx mori silkworm cocoons, to provide solutions for human platelet production (Di Buduo et al. Blood 2015). Here, we aimed at exploiting this technology by manufacturing innovative 3D silk-based bone marrow niches and flow chambers to direct the generation of compartmentalized erythroblastic islands and set effective protocols to produce fully mature human erythrocytes ex vivo. Methods We developed an all-natural tissue model consisting of silk fibroin and extracellular matrix proteins. The spongy scaffold was enclosed in a protective chamber providing a dynamic flow of nutrients and cytokines through an interconnected vascular-like network. The system was cultured for up to 3 weeks with hematopoietic stem and progenitor cells (HSPCs) from human mobilized peripheral blood, upon informed consent and in accordance with the Declaration of Helsinki. Results HSPCs could be efficiently differentiated into erythroblasts under the control of a bespoke cocktail of cytokines in combination with increasing concentrations of transferrin. After 1 week of culture, flow cytometry analysis demonstrated the gradual loss of stem-associated markers, such as CD34, CD105, and CD117, and the acquisition of the lineage-specific markers CD71 (transferrin receptor), CD36 (a transmembrane scavenger receptor), and CD235a (glycophorin-α). The analysis of the spatial distribution of cells within the silk scaffold, by real-time imaging, demonstrated the spontaneous development of cellular nests, resembling the physiological structure of the erythroblastic islands typically, but not exclusively, aggregating around and/or next to co-cultured CD68+ macrophages. After 2 weeks of differentiation, the population turned to a CD36-CD71lowCD235ahigh profile, indicative of terminal erythroblast maturation. Starting from week 3, fully mature CD36-C71-CD235ahigh erythrocytes were mainly localized at the periphery of the erythroblastic islands, in close contact with macrophages. Dynamic perfusion of the system at physiologically relevant shear stress allowed the collection of >90% enucleated CD235ahigh erythrocytes. Cells were fully hemoglobinized and expressed both α- and β-globin. May-Grunwald Giemsa staining revealed that the size, shape, and color of cultured erythrocytes were perfectively comparable to donor cells. The recovered erythrocytes showed significant downregulation of the expression of α4β1 integrin, which was retained by immature erythroblasts adhering to the silk scaffolds. Conclusion Our data demonstrated that the spatiotemporal control of the dynamics of cell-cell and cell-matrix interaction, together, with controllable shear forces, within a 3D physiologically relevant microenvironment, may be the key to guiding efficient erythropoiesis ex vivo. The silk bone marrow model represents a unique sustainable resource to gain insights into mechanisms of human erythropoiesis, and advance basic research to robust clinical translation.

A 3D-induced pluripotent stem cell-derived human neural culture model to study certain molecular and biochemical aspects of Alzheimer’s disease

PurposeAlzheimer’s disease (AD) early pathology needs better understanding and models. Here, we describe a human induced pluripotent stem cells (iPSCs)-derived 3D neural culture model to study certain aspects of AD biochemistry and pathology.MethodiPSCs derived from controls and AD patients with Presenilin1 mutations were cultured in a 3D platform with a similar microenvironment to the brain, to differentiate into neurons and astrocytes and self-organise into 3D structures by 3 weeks of differentiation in vitro.ResultsCells express astrocytic (GFAP), neuronal (β3-Tubulin, MAP2), glutamatergic (VGLUT1), GABAergic (GAD65/67), pre-synaptic (Synapsin1) markers and a low level of neural progenitor cell (Nestin) marker after 6 and 12 weeks of differentiation in 3D. The foetal 3R Tau isoforms and adult 4R Tau isoforms were detected at 6 weeks post differentiation, showing advanced neuronal maturity. In the 3D AD cells, total and insoluble Tau levels were higher than in 3D control cells.ConclusionOur data indicates that this model may recapitulate the early biochemical and pathological disease features and can be a relevant platform for studying early cellular and biochemical changes and the identification of drug targets.

Dynamics of Metabolic Pathways and Stress Response Patterns during Human Neural Stem Cell Proliferation and Differentiation.

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1–NRF2–ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.

Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity

Oxidative stress is caused by an imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS). This imbalance plays an important role in brain aging and age-related neurodegenerative diseases. In the context of Parkinson's disease (PD), the sensitivity of dopaminergic neurons in the substantia nigra pars compacta to oxidative stress is considered a key factor of PD pathogenesis. Here we study the effect of different oxidative stress-inducing compounds (6-OHDA, MPTP or MPP+) on the population of dopaminergic neurons in an iPSC-derived human brain 3D model (aka BrainSpheres). Treatment with 6-OHDA, MPTP or MPP+ at 4 weeks of differentiation disrupted the dopaminergic neuronal phenotype in BrainSpheres at (50, 5000, 1000 μM respectively). 6-OHDA increased ROS production and decreased mitochondrial function most efficiently. It further induced the greatest changes in gene expression and metabolites related to oxidative stress and mitochondrial dysfunction. Co-culturing BrainSpheres with an endothelial barrier using a transwell system allowed the assessment of differential penetration capacities of the tested compounds and the damage they caused in the dopaminergic neurons within the BrainSpheres In conclusion, treatment with compounds known to induce PD-like phenotypes in vivo caused molecular deficits and loss of dopaminergic neurons in the BrainSphere model. This approach therefore recapitulates common animal models of neurodegenerative processes in PD at similarly high doses. The relevance as tool for drug discovery is discussed.

Ex Vivo Production of Large Numbers of Genetically Modified NK Cells from Cord Blood or Mobilized Peripheral Blood CD34 + Cells Using Notch Ligand Delta-like 4 Culture System

Natural killer (NK) cells are an essential component of human innate immune, with a remarkable ability to provide protection against cancer and viral infections. NK-based immune therapy has several advantages over T cell immunotherapy: NK cells do not cause graft-versus-host disease and do not induce T-cell driven inflammatory cytokine storm. Moreover, unlike T cell, NK cells do not need prior sensitization, specific antigen recognition and clonal expansion for their cytotoxic effector functions and they can rapidly trigger cytotoxicity against their targets. NK cells can be expanded from multiple sources including peripheral blood mononuclear cells (PBMCs), umbilical cord blood (UCB), or mobilized peripheral blood (mPB) CD34 + cells. Yet the lack of efficient methods for in-vitro NK cell expansion, purification and genetic modification limits the clinical use of NK cell therapy.We aimed at developing a simple, scalable and clinically feasible technique for therapeutic NK cell production based on our recently published culture system that can produce large numbers of unmodified or genetically modified CD7 + T lymphoid progenitors from UCB and mPB CD34 + cells in 7 days on immobilized Notch-ligand delta-like 4 recombinant protein DLL4. Since T cells and NK cells share a common T/NK cell progenitor, we are interested to investigate the NK cell potential of DLL4 culture-generated T lymphoid progenitors.Firstly, we tested the NK cell potential of the transduced or non- transduced UCB or mPB CD34 +-derived progenitors from DLL4 culture by subjecting them to a feeder free NK cell differentiation for 3 weeks. HSPC-derived progenitors can be efficiently transduced with lentiviral vectors (average transduction efficacy: 50%). The transduced/ non-transduced progenitors were able to efficiently differentiate into NK (CD3 -CD56 +) cells beginning from one week of differentiation, reaching a frequencies of NK cells of >90% without any detectable T cell contamination at week 2. The phenotypic characterization of the NK cells demonstrates the presence of activation receptors such as NKG2D, DNAM-1, NKp30, NKp44 and NKp46 while lacking the inhibitory receptors like KLRG1, KIR2DL2/DL3 and KIR3DL1/DL2. Interestingly, these cells express the transcription factors known to be essential for NK cell differentiation and functions such as Eomes, T-bet and ID2. Additionally, the CB or mPB HSPC-derived NK cells (both transduced/ non-transduced) express perforin and granzyme B reflecting their ability to show cytotoxic potential.Further, the stimulation of UCB or mPB derived NK cells with myelogenous leukemia cell line K562 cells showed high level of degranulation, which is the key step for Interferon gamma (IFNg) induction. An analysis cytotoxic activity of the CB or mPB derived NK cells against K562 cells and monocytic leukemia cell line THP1 cells showed their ability to efficiently kill K562 cells compared to THP1 cells, however the UCB derived NK cells were highly cytotoxic compared to mPB derived NK cells.These data suggests that our DLL4 culture system, along with feeder-free NK cell differentiation is a unique combination that is able to give rise to high number of pure NK cell population with high cytotoxic potential. These results lay a foundation towards an easier approach to NK cell therapy for effective treatment of cancers and viral infections, which will be developed in collaboration with Smart Immune Inc. DisclosuresCavazzana: Smart Immune: Other: co-founder.

Autism in Three Dimensions: Using Brain Organoids to Study Potential Gene-Environment Interactions.

Autism in Three Dimensions: Using Brain Organoids to Study Potential Gene-Environment Interactions.

Growth and mineralization of osteoblasts from mesenchymal stem cells on microporous membranes: Epithelial-like growth with transmembrane resistance and pH gradient

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 μm pores at a density of (2 ± 0.4) x 106 pores per cm2. Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ∼1400 Ω•cm2 at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Human iPS cells engender corneal epithelial stem cells with holoclone-forming capabilities

SummaryHuman induced pluripotent stem cells (hiPSCs) can generate a multiplicity of organoids, yet no compelling evidence currently exists as to whether or not these contain tissue-specific, holoclone-forming stem cells. Here, we show that a subpopulation of cells in a hiPSC-derived corneal epithelial cell sheet is positive for ABCB5 (ATP-binding cassette, sub-family B, member 5), a functional marker of adult corneal epithelial stem cells. These cells possess remarkable holoclone-forming capabilities, which can be suppressed by an antibody-mediated ABCB5 blockade. The cell sheets are generated from ABCB5+ hiPSCs that first emerge in 2D eye-like organoids around six weeks of differentiation and display corneal epithelial immunostaining characteristics and gene expression patterns, including sustained expression of ABCB5. The findings highlight the translational potential of ABCB5-enriched, hiPSC-derived corneal epithelial cell sheets to recover vision in stem cell-deficient human eyes and represent the first report of holoclone-forming stem cells being directly identified in an hiPSC-derived organoid.