Transcription Factor-Mediated Generation of Dopaminergic Neurons from Human iPSCs-A Comparison of Methods.

Dopaminergic neurons are the predominant brain cells affected in Parkinson's disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson's disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons.

In situ biomineralization reinforcing anisotropic nanocellulose scaffolds for guiding the differentiation of bone marrow-derived mesenchymal stem cells.

Nanocellulose (NC) is a promising biopolymer for various biomedical applications owing to its biocompatibility and low toxicity. However, it faces challenges in tissue engineering (TE) applications due to the inconsistency of the microenvironment within the NC-based scaffolds with target tissues, including anisotropy microstructure and biomechanics. To address this challenge, a facile swelling-induced nanofiber alignment and a novel in situ biomineralization reinforcement strategies were developed for the preparation of NC-based scaffolds with tunable anisotropic structure and mechanical strength for guiding the differentiation of bone marrow-derived mesenchymal stem cells for potential TE application. The bacterial cellulose (BC) and cellulose nanofibrils (CNFs) based scaffolds with tunable swelling anisotropic index in the range of 10-100 could be prepared by controlling the swelling medium. The in situ biomineralization efficiently reinforced the scaffolds with 2-4 times and 10-20 times modulus increasement for BC and CNFs, respectively. The scaffolds with higher mechanical strength were superior in supporting cell growth and proliferation, suggesting the potential application in TE application. This work demonstrated the feasibility of the proposed strategy in the preparation of scaffolds with mechanical anisotropy to induce cells-directed differentiation for TE applications.

Generation of human alveolar epithelial type I cells from pluripotent stem cells.

Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here, we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, enriched in these cells. Next, we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s.

Human pluripotent stem cell (hPSC)-derived microglia for the study of brain disorders. A comprehensive review of existing protocols.

Microglia, resident immune cells of the brain that originate from the yolk sac, play a critical role in maintaining brain homeostasis by monitoring and phagocytosing pathogens and cellular debris in the central nervous system (CNS). While they share characteristics with myeloid cells, they are distinct from macrophages. In response to injury, microglia release pro-inflammatory factors and contribute to brain homeostasis through activities such as synapse pruning and neurogenesis. To better understand their role in neurological disorders, the generation of in vitro models of human microglia has become essential. These models, derived from patient-specific induced pluripotent stem cells (iPSCs), provide a controlled environment to study the molecular and cellular mechanisms underlying microglia-mediated neuroinflammation and neurodegeneration. The incorporation or generation of microglia into three-dimensional (3D) organoid cultures provides a more physiologically relevant environment that offers further opportunities to study microglial dynamics and disease modeling. This review describes several protocols that have been recently developed for the generation of human-induced microglia. Importantly, it highlights the promise of these in vitro models in advancing our understanding of brain disorders and facilitating personalized drug screening.

Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders.

Human-induced pluripotent stem cell (hiPSC) technology has enabled comprehensive human cell-based disease modeling in vitro. Due to limited accessibility of primary human neurons as well as species-specific divergence between human and rodent brain tissues, hiPSC-derived neurons have become a popular tool for studying neuronal biology in a dish. Here, we provide methods for transcription factor-driven directed differentiation of neurons from hiPSCs via a neural progenitor cell (NPC) intermediate. Doxycycline-inducible expression of neuron fate-determining transcription factors neurogenin 2 (NGN2) and achaete-scute homolog 1 (ASCL1) enables rapid and controllable differentiation of human neurons for disease modeling applications. The provided method is also designed to improve the reproducibility of human neuron differentiation by reducing the batch-to-batch variation of NPC differentiation and lentiviral transduction.

Making Human Hematopoietic Stem Cells Without Transgenes.

Creating hematopoietic stem cells (HSCs) capable of multilineage engraft while possessing the ability to self-renew stands as a pivotal achievement within the field of regenerative medicine. However, achieving the generation of these cells without transgene expression or teratoma formation has not been fully accomplished. In a recent publication featured in Cell Stem Cell, Piau et al. document the production of functional HSCs derived from human-induced pluripotent stem cells (hiPSCs). They achieved this through a one-step differentiation protocol that notably does not require any transgene expression. hiPSCs-derived HSCs can engraft and self-renew upon serial transplantation and they are able to reconstitute lymphoid, myeloid, and erythroid compartments. This study presents a promising system to further study human HSC ontogeny, and it might represent a crucial step to obtain HSCs.

iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia.

Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.

3D Hydrogel Encapsulation Regulates Nephrogenesis in Kidney Organoids.

Stem cell-derived kidney organoids contain nephron segments that recapitulate morphological and functional aspects of the human kidney. However, directed differentiation protocols for kidney organoids are largely conducted using biochemical signals to control differentiation. Here, the hypothesis that mechanical signals regulate nephrogenesis is investigated in 3D culture by encapsulating kidney organoids within viscoelastic alginate hydrogels with varying rates of stress relaxation. Tubular nephron segments are significantly more convoluted in kidney organoids differentiated in encapsulating hydrogels when compared with those in suspension culture. Hydrogel viscoelasticity regulates the spatial distribution of nephron segments within the differentiating kidney organoids. Consistent with these observations, a particle-based computational model predicts that the extent of deformation of the hydrogel-organoid interface regulates the morphology of nephron segments. Elevated extracellular calcium levels in the culture medium, which can be impacted by the hydrogels, decrease the glomerulus-to-tubule ratio of nephron segments. These findings reveal that hydrogel encapsulation regulates nephron patterning and morphology and suggest that the mechanical microenvironment is an important design variable for kidney regenerative medicine.

Modeling Human Paraxial Mesoderm Development with Pluripotent Stem Cells.

Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites are sequentially formed from head to tail in a rhythmic manner controlled by an oscillating gene regulatory network known as the segmentation clock. We know very little about this important process during human development due to limited access to human embryos and ethical concerns. To bypass these difficulties, model systems derived from human pluripotent stem cells have been established. Here, we detail three protocols modeling different aspects of human paraxial mesoderm development in vitro: a 2D cell monolayer system recapitulating dynamics of the human segmentation clock, a 3D organoid system called "somitoid" supporting the simultaneous formation of somite-like structures, and another organoid system called "segmentoid" reconstituting in vivo-like hallmarks of somitogenesis. Together, these complementary model systems provide an excellent platform to decode somitogenesis and advance human developmental biology.

Preparation of astrocytes by directed differentiation of pluripotent stem cells and somatic cell transdifferentiation.

Astrocytes (ACs) are the most widely distributed cells in the mammalian central nervous system, which are essential for the function and homeostasis of nervous system. Increasing evidence indicates that ACs also participate in the development of many neurological diseases and repair after nerve injury. ACs cultured in vitro provide a cellular model for studying astrocytic development, function, and the pathogenesis of associated diseases. The preparation of primary ACs (pACs) faces many limitations, so it is important to obtain high-quality ACs by the differentiation of pluripotent stem cell (PSC) or somatic cell transdifferentiation. Initially, researchers mainly tried to induce embryonic stem cells to differentiate into ACs via embryoid body (EB) and then turned to employ induced PSCs as seed cells to explore more simple and efficient directed differentiation strategies, and serum-free culture was delved to improve the quality of induced ACs. While exploring the induction of ACs by the overexpression of AC-specific transcription factors, researchers also began to investigate small molecule-mediated somatic cell transdifferentiation. Here, we provide an updated review on the research progresses in this field.

Reconstructing human brown fat developmental trajectory in vitro.

Brown adipocytes (BAs) represent a specialized cell type that is able to uncouple nutrient catabolism from ATP generation to dissipate energy as heat. In humans, the brown fat tissue is composed of discrete depots found throughout the neck and trunk region. BAs originate from a precursor common to skeletal muscle, but their developmental trajectory remains poorly understood. Here, we used single-cell RNA sequencing to characterize the development of interscapular brown fat in mice. Our analysis identified a transient stage of BA differentiation characterized by the expression of the transcription factor GATA6. We show that recapitulating the sequence of signaling cues identified in mice can lead to efficient differentiation of BAs in vitro from human pluripotent stem cells. These precursors can in turn be efficiently converted into functional BAs that can respond to signals mimicking adrenergic stimuli by increasing their metabolism, resulting in heat production.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Airway stem cell reconstitution by the transplantation of primary or pluripotent stem cell-derived basal cells.

Life-long reconstitution of a tissue's resident stem cell compartment with engrafted cells has the potential to durably replenish organ function. Here, we demonstrate the engraftment of the airway epithelial stem cell compartment via intra-airway transplantation of mouse or human primary and pluripotent stem cell (PSC)-derived airway basal cells (BCs). Murine primary or PSC-derived BCs transplanted into polidocanol-injured syngeneic recipients give rise for at least two years to progeny that stably display the morphologic, molecular, and functional phenotypes of airway epithelia. The engrafted basal-like cells retain extensive self-renewal potential, evident by the capacity to reconstitute the tracheal epithelium through seven generations of secondary transplantation. Using the same approach, human primary or PSC-derived BCs transplanted into NOD scid gamma (NSG) recipient mice similarly display multilineage airway epithelial differentiation in vivo. Our results may provide a step toward potential future syngeneic cell-based therapy for patients with diseases resulting from airway epithelial cell damage or dysfunction.

Durable alveolar engraftment of PSC-derived lung epithelial cells into immunocompetent mice.

Durable reconstitution of the distal lung epithelium with pluripotent stem cell (PSC) derivatives, if realized, would represent a promising therapy for diseases that result from alveolar damage. Here, we differentiate murine PSCs into self-renewing lung epithelial progenitors able to engraft into the injured distal lung epithelium of immunocompetent, syngeneic mouse recipients. After transplantation, these progenitors mature in the distal lung, assuming the molecular phenotypes of alveolar type 2 (AT2) and type 1 (AT1) cells. After months in vivo, donor-derived cells retain their mature phenotypes, as characterized by single-cell RNA sequencing (scRNA-seq), histologic profiling, and functional assessment that demonstrates continued capacity of the engrafted cells to proliferate and differentiate. These results indicate durable reconstitution of the distal lung's facultative progenitor and differentiated epithelial cell compartments with PSC-derived cells, thus establishing a novel model for pulmonary cell therapy that can be utilized to better understand the mechanisms and utility of engraftment.

Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols.

Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.

Differentiation of neurosphere after transplantation into the damaged spinal cord.

This study aimed to compare the differentiation and survival of human neural stem/progenitor cells of various origins in vitro and after transplantation into the injured spinal cord of laboratory animals. Rats with simulated spinal cord injury were transplanted with neurosphere cells obtained by directed differentiation of HUES6 cell lines. Fluorescence microscopy was used to visualize the obtained results. HUES6#1 and iPSC#1 neurospheres showed a wide range of markers associated with glial differentiation. The expression of the proliferation marker Ki67 did not exceed 25%, both in the lines of early and late neurospheres. Although neurospheres did not fully differentiate into astrocytes in vitro, they massively approached the GFAP+ astrocyte phenotype when exposed to the transplanted environment. PSC-derived neurospheres transplanted into the site of SM injury without additional growth factors showed only moderate survival, a significant degree of differentiation into astrocytes, and moderate differentiation into neurons. The difference in the survival and differentiation of HUES6#1 and iPSC#1 neurospheres, both in vitro and in vivo, can be explained by the difference in the regulatory behavior of signaling molecules corresponding to the source of origin of PSCs. Derivatives of human PSCs of various origins obtained according to the described differentiation protocol did not mature into astrocytic populations, nor did the glycogenic transition of PSC-derived NSCs occur in vitro. The study demonstrated the impact of the injured spinal cord microenvironment on the differentiation of transplanted HUES6#1 and iPSC#1 into astrocytes. The results showed that HUES6-derived neurospheres generated 90% of GFAP+ astrocytes and 5-10% of early neurons, while iPSC-derived neurospheres generated an average of 74% GFAP+ astrocytes and 5% of early neurons in vivo.

SIX1+PAX3+ identify a progenitor for myogenic lineage commitment from hPSCs.

The earliest skeletal muscle progenitor cells (SMPCs) derived from human pluripotent stem cells (hPSCs) are often identified by factors expressed by a diverse number of progenitors. An early transcriptional checkpoint that defines myogenic commitment could improve hPSC differentiation to skeletal muscle. Analysis of several myogenic factors in human embryos and early hPSC differentiations found SIX1+PAX3+ co-expression was most indictive of myogenesis. Using dCas9-KRAB hPSCs, we demonstrate that early inhibition of SIX1 alone significantly decreased PAX3 expression, reduced PAX7+ SMPCs, and myotubes later in differentiation. Emergence of SIX1+PAX3+ precursors can be improved by manipulating seeding density, monitoring metabolic secretion and altering the concentration of CHIR99021. These modifications resulted in the co-emergence of hPSC-derived sclerotome, cardiac and neural crest that we hypothesized enhanced hPSC myogenic differentiation. Inhibition of non-myogenic lineages modulated PAX3 independent of SIX1. To better understand SIX1 expression, we compared directed differentiations to fetal progenitors and adult satellite cells by RNA-seq. Although SIX1 continued to be expressed across human development, SIX1 co-factor expression was dependent on developmental timing. We provide a resource to enable efficient derivation of skeletal muscle from hPSCs.

Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.

IPSC-Derived Sensory Neurons Directing Fate Commitment of Human BMSC-Derived Schwann Cells: Applications in Traumatic Neural Injuries.

The in vitro derivation of Schwann cells from human bone marrow stromal cells (hBMSCs) opens avenues for autologous transplantation to achieve remyelination therapy for post-traumatic neural regeneration. Towards this end, we exploited human induced pluripotent stem-cell-derived sensory neurons to direct Schwann-cell-like cells derived from among the hBMSC-neurosphere cells into lineage-committed Schwann cells (hBMSC-dSCs). These cells were seeded into synthetic conduits for bridging critical gaps in a rat model of sciatic nerve injury. With improvement in gait by 12-week post-bridging, evoked signals were also detectable across the bridged nerve. Confocal microscopy revealed axially aligned axons in association with MBP-positive myelin layers across the bridge in contrast to null in non-seeded controls. Myelinating hBMSC-dSCs within the conduit were positive for both MBP and human nucleus marker HuN. We then implanted hBMSC-dSCs into the contused thoracic cord of rats. By 12-week post-implantation, significant improvement in hindlimb motor function was detectable if chondroitinase ABC was co-delivered to the injured site; such cord segments showed axons myelinated by hBMSC-dSCs. Results support translation into a protocol by which lineage-committed hBMSC-dSCs become available for motor function recovery after traumatic injury to both peripheral and central nervous systems.

Endothelial progenitor cells derived from embryonic stem cells prevent alveolar simplification in a murine model of bronchopulmonary dysplasia.

Introduction: Vascular remodeling and compromised alveolar development are hallmarks of chronic pulmonary diseases such as bronchopulmonary dysplasia (BPD). Despite advances in neonatal healthcare the number of BPD cases worldwide continues to increase. One approach to overcoming the premature arrest in lung development seen in BPD is to stimulate neonatal angiogenesis via delivery and engraftment of endothelial progenitor cells (EPCs). One such population is resident to the pulmonary microvasculature and expresses both FOXF1 and c-KIT. Previous studies have shown that c-KIT+FOXF1+ EPCs are highly sensitive to elevated levels of oxygen (hyperoxia) and are decreased in premature infants with BPD and hyperoxia-induced BPD mouse models. We hypothesize that restoring EPCs through transplantation of c-KIT+FOXF1+ EPCs derived in vitro from pluripotent embryonic stem cells (ESCs), will stimulate neonatal angiogenesis and alveolarization in mice with hyperoxia-induced lung injury. Methods: Utilizing a novel ESC line with a FOXF1:GFP reporter, we generated ESC-derived c-KIT+FOXF1+ EPCs in vitro. Using a second ESC line which contains FOXF1:GFP and tdTomato transgenes, we differentiated ESCs towards c-KIT+FOXF1+ EPCs and tracked them in vivo after injection into the neonatal circulation of hyperoxia-injured mice. After a recovery period in room air conditions, we analyzed c-KIT+FOXF1+ EPC engraftment and quantified the number of resident and circulating endothelial cells, the size of alveolar spaces, and the capillary density after EPC transplantations. Results and conclusion: Herein, we demonstrate that addition of BMP9 to the directed endothelial differentiation protocol results in very efficient generation of c-KIT+FOXF1+ EPCs from pluripotent ESCs. ESC-derived c-KIT+FOXF1+ EPCs effectively engraft into the pulmonary microvasculature of hyperoxia-injured mice, promote vascular remodeling in alveoli, increase the number of resident and circulating endothelial cells, and improve alveolarization. Altogether, these results provide a proof-of-principle that cell therapy with ESC-derived c-KIT+FOXF1+ EPCs can prevent alveolar simplification in a hyperoxia-induced BPD mouse model.