The Breakthroughs and Caveats of Using Human Pluripotent Stem Cells in Modeling Alzheimer's Disease.

Modeling Alzheimer's disease (AD) using human-induced pluripotent stem cells (iPSCs) is a field now spanning 15 years. Developments in the field have shown a shift in using simple 2D cortical neuron models to more advanced tri-cultures and 3D cerebral organoids that recapitulate more features of the disease. This is largely due to development and optimization of new cell protocols. In this review, we highlight recent major breakthroughs in the AD field and the implications this has in modeling AD using iPSCs (AD-iPSCs). To date, AD-iPSCs have been largely used to recapitulate and study impaired amyloid precursor protein (APP) processing and tau phosphorylation in both familial and sporadic AD. AD-iPSCs have also been studied for varying neuronal and glial dysfunctions. Moreover, they have been useful for discovering new molecular mechanisms, such as identifying proteins that bridge APP processing with tau phosphorylation and for identifying molecular pathways that bridge APP processing dysfunction with impaired cholesterol biosynthesis. Perhaps the greatest use of AD-iPSCs has been in discovering compounds via drug screening, that reduce amyloid beta (Aß) in neurons, such as the anti-inflammatory compound, cromolyn, and antiparasitic drugs, avermectins. In addition, high content screening using AD-iPSCs has led to the identification of statins that can reduce levels of phosphorylated tau (p-Tau) in neurons. Some of these compounds have made it through to testing in human clinical trials. Improvements in omic technologies including single cell RNA sequencing and proteomics as well as advances in production of iPSC-cerebral organoids and tri-cultures is likely to result in the further discovery of new drugs and treatments for AD. Some caveats remain in the field, including, long experimental conditions to create mature neurons, high costs of media that limit research capabilities, and a lack of reproducibility using current iPSC-cerebral organoid protocols. Despite these current limitations, AD-iPSCs remain an excellent cellular model for studying AD mechanisms and for drug discovery.

Production of human entorhinal stellate cell-like cells by forward programming shows an important role of Foxp1 in reprogramming.

Stellate cells are principal neurons in the entorhinal cortex that contribute to spatial processing. They also play a role in the context of Alzheimer's disease as they accumulate Amyloid beta early in the disease. Producing human stellate cells from pluripotent stem cells would allow researchers to study early mechanisms of Alzheimer's disease, however, no protocols currently exist for producing such cells. In order to develop novel stem cell protocols, we characterize at high resolution the development of the porcine medial entorhinal cortex by tracing neuronal and glial subtypes from mid-gestation to the adult brain to identify the transcriptomic profile of progenitor and adult stellate cells. Importantly, we could confirm the robustness of our data by extracting developmental factors from the identified intermediate stellate cell cluster and implemented these factors to generate putative intermediate stellate cells from human induced pluripotent stem cells. Six transcription factors identified from the stellate cell cluster including RUNX1T1, SOX5, FOXP1, MEF2C, TCF4, EYA2 were overexpressed using a forward programming approach to produce neurons expressing a unique combination of RELN, SATB2, LEF1 and BCL11B observed in stellate cells. Further analyses of the individual transcription factors led to the discovery that FOXP1 is critical in the reprogramming process and omission of RUNX1T1 and EYA2 enhances neuron conversion. Our findings contribute not only to the profiling of cell types within the developing and adult brain's medial entorhinal cortex but also provides proof-of-concept for using scRNAseq data to produce entorhinal intermediate stellate cells from human pluripotent stem cells in-vitro.

Prenatal development of the human entorhinal cortex.

Little is known about the development of the human entorhinal cortex (EC), a major hub in a widespread network for learning and memory, spatial navigation, high-order processing of object information, multimodal integration, attention and awareness, emotion, motivation, and perception of time. We analyzed a series of 20 fetal and two adult human brains using Nissl stain, acetylcholinesterase (AChE) histochemistry, and immunocytochemistry for myelin basic protein (MBP), neuronal nuclei antigen (NeuN), a pan-axonal neurofilament marker, and synaptophysin, as well as postmortem 3T MRI. In comparison with other parts of the cerebral cortex, the cytoarchitectural differentiation of the EC begins remarkably early, in the 10th week of gestation (w.g.). The differentiation occurs in a superficial magnocellular layer in the deep part of the marginal zone, accompanied by cortical plate (CP) condensation and multilayering of the deep part of CP. These processes last until the 13-14th w.g. At 14 w.g., the superficial lamina dissecans (LD) is visible, which divides the CP into the lamina principalis externa (LPE) and interna (LPI). Simultaneously, the rostral LPE separates into vertical cell-dense islands, whereas in the LPI, the deep LD emerges as a clear acellular layer. In the 16th w.g., the LPE remodels into vertical cell-dense and cell-sparse zones with a caudorostral gradient. At 20 w.g., NeuN immunoreactivity is most pronounced in the islands of layer II cells, whereas migration and differentiation inside-out gradients are seen simultaneously in both the upper (LPE) and the lower (LPI) pyramidal layers. At this stage, the EC adopts for the first time an adult-like cytoarchitectural organization, the superficial LD becomes discernible by 3T MRI, MBP-expressing oligodendrocytes first appear in the fimbria and the perforant path (PP) penetrates the subiculum to reach its molecular layer and travels along through the Cornu Ammonis fields to reach the suprapyramidal blade of the dentate gyrus, whereas the entorhinal-dentate branch perforates the hippocampal sulcus about 2-3 weeks later. The first AChE reactivity appears as longitudinal stripes at 23 w.g. in layers I and II of the rostrolateral EC and then also as AChE-positive in-growing fibers in islands of superficial layer III and layer II neurons. At 40 w.g., myelination of the PP starts as patchy MBP-immunoreactive oligodendrocytes and their processes. Our results refute the possibility of an inside-out pattern of the EC development and support the key role of layer II prospective stellate cells in the EC lamination. As the early cytoarchitectural differentiation of the EC is paralleled by the neurochemical development, these developmental milestones in EC structure and connectivity have implications for understanding its normal function, including its puzzling modular organization and potential contribution to consciousness content (awareness), as well as for its insufficiently explored deficits in developmental, psychiatric, and degenerative brain disorders.

Development of the Entorhinal Cortex Occurs via Parallel Lamination During Neurogenesis.

The entorhinal cortex (EC) is the spatial processing center of the brain and structurally is an interface between the three layered paleocortex and six layered neocortex, known as the periarchicortex. Limited studies indicate peculiarities in the formation of the EC such as early emergence of cells in layers (L) II and late deposition of LIII, as well as divergence in the timing of maturation of cell types in the superficial layers. In this study, we examine developmental events in the entorhinal cortex using an understudied model in neuroanatomy and development, the pig and supplement the research with BrdU labeling in the developing mouse EC. We determine the pig serves as an excellent anatomical model for studying human neurogenesis, given its long gestational length, presence of a moderate sized outer subventricular zone and early cessation of neurogenesis during gestation. Immunohistochemistry identified prominent clusters of OLIG2+ oligoprogenitor-like cells in the superficial layers of the lateral EC (LEC) that are sparser in the medial EC (MEC). These are first detected in the subplate during the early second trimester. MRI analyses reveal an acceleration of EC growth at the end of the second trimester. BrdU labeling of the developing MEC, shows the deeper layers form first and prior to the superficial layers, but the LV/VI emerges in parallel and the LII/III emerges later, but also in parallel. We coin this lamination pattern parallel lamination. The early born Reln+ stellate cells in the superficial layers express the classic LV marker, Bcl11b (Ctip2) and arise from a common progenitor that forms the late deep layer LV neurons. In summary, we characterize the developing EC in a novel animal model and outline in detail the formation of the EC. We further provide insight into how the periarchicortex forms in the brain, which differs remarkably to the inside-out lamination of the neocortex.

A Comparative Assessment of Marker Expression Between Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells and the Developing Pig Heart.

The course of differentiation of pluripotent stem cells into cardiomyocytes and the intermediate cell types are characterized using molecular markers for different stages of development. These markers have been selected primarily from studies in the mouse and from a limited number of human studies. However, it is not clear how well mouse cardiogenesis compares with human cardiogenesis at the molecular level. We tackle this issue by analyzing and comparing the expression of common cardiomyogenesis markers [platelet-derived growth factor receptor, alpha polypeptide (PDGFR-α), fetal liver kinase 1 (FLK1), ISL1, NK2 homeobox 5 (NKX2.5), cardiac troponin T (CTNT), connexin43 (CX43), and myosin heavy chain 7 (MYHC-B)] in the developing pig heart at embryonic day (E)15, E16, E18, E20, E22, and E24 and in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs). We found that porcine expression of the mesoderm marker FLK1 and the cardiac progenitor marker ISL1 was in line with our differentiating hiPSC and reported murine expression. The cardiac lineage marker NKX2.5 was expressed at almost all stages in the pig and hiPSC, with an earlier onset in the hiPSC compared with reported murine expression. Markers of immature cardiomyocytes, CTNT, and MYHC-B were consistently expressed throughout E16-E70 in the pig, which is comparable with mouse development, whereas the markers increased over time in the hiPSC. However, the commonly used mature cardiomyocyte marker, CX43, should be used with caution, as it was also expressed in the pig mesoderm, as well as hiPSC immature cardiomyocytes, while this has not been reported in mice. Based on our observations in the various species, we suggest to use FLK1/PDGFR-α for identifying cardiac mesoderm and ISL1/NKX2.5 for cardiac progenitors. Furthermore, a combination of two or more of the following, CTNT+/MYHC-B+/ISL1+ could mark immature cardiomyocytes and CTNT+/ISL1- mature cardiomyocytes. CX43 should be used together with sarcomeric proteins. This knowledge may help improving differentiation of hiPSC into more in vivo-like cardiac tissue in the future.

A community-based transcriptomics classification and nomenclature of neocortical cell types.

To understand the function of cortical circuits, it is necessary to catalog their cellular diversity. Past attempts to do so using anatomical, physiological or molecular features of cortical cells have not resulted in a unified taxonomy of neuronal or glial cell types, partly due to limited data. Single-cell transcriptomics is enabling, for the first time, systematic high-throughput measurements of cortical cells and generation of datasets that hold the promise of being complete, accurate and permanent. Statistical analyses of these data reveal clusters that often correspond to cell types previously defined by morphological or physiological criteria and that appear conserved across cortical areas and species. To capitalize on these new methods, we propose the adoption of a transcriptome-based taxonomy of cell types for mammalian neocortex. This classification should be hierarchical and use a standardized nomenclature. It should be based on a probabilistic definition of a cell type and incorporate data from different approaches, developmental stages and species. A community-based classification and data aggregation model, such as a knowledge graph, could provide a common foundation for the study of cortical circuits. This community-based classification, nomenclature and data aggregation could serve as an example for cell type atlases in other parts of the body.

Isolation and culture of porcine primary fetal progenitors and neurons from the developing dorsal telencephalon.

The development of long-term surviving fetal cell cultures from primary cell tissue from the developing brain is important for facilitating studies investigating neural development and for modelling neural disorders and brain congenital defects. The field faces current challenges in co-culturing both progenitors and neurons long-term. Here, we culture for the first time, porcine fetal cells from the dorsal telencephalon at embryonic day (E) 50 and E60 in conditions that promoted both the survival of progenitor cells and young neurons. We applied a novel protocol designed to collect, isolate and promote survival of both progenitors and young neurons. Herein, we used a combination of low amount of fetal bovine serum, together with pro-survival factors, including basic fibroblast growth factor and retinoic acid, together with arabinofuranosylcytosine and could maintain progenitors and facilitate in vitro differentiation into calbindin 1+ neurons and reelin+ interneurons for a period of 7 days. Further improvements to the protocol that might extend the survival of the fetal primary neural cells would be beneficial. The development of new porcine fetal culture methods is of value for the field, given the pig's neuroanatomical and developmental similarities to the human brain.

Evidence for nucleolar dysfunction in Alzheimer's disease.

The nucleolus is a dynamically changing organelle that is central to a number of important cellular functions. Not only is it important for ribosome biogenesis, but it also reacts to stress by instigating a nucleolar stress response and is further involved in regulating the cell cycle. Several studies report nucleolar dysfunction in Alzheimer's disease (AD). Studies have reported a decrease in both total nucleolar volume and transcriptional activity of the nucleolar organizing regions. Ribosomes appear to be targeted by oxidation and reduced protein translation has been reported. In addition, several nucleolar proteins are dysregulated and some of these appear to be implicated in classical AD pathology. Some studies also suggest that the nucleolar stress response may be activated in AD, albeit this latter research is rather limited and requires further investigation. The purpose of this review is to draw the connections of all these studies together and signify that there are clear changes in the nucleolus and the ribosomes in AD. The nucleolus is therefore an organelle that requires more attention than previously given in relation to understanding the biological mechanisms underlying the disease.

Generation of transgene-free porcine intermediate type induced pluripotent stem cells.

Physiologically and anatomically, humans and pigs share many similarities, which make porcine induced pluripotent stem cells (piPSCs) very attractive for modeling human cell therapy as well as for testing safety of iPSC based cell replacement therapies. To date, several integrative and non-integrative strategies have been reported to successfully generate piPSCs, but all resulting piPSCs had integration of transgenes. The use of integrative methods has the disadvantage of potential lack of silencing or inappropriate re-activation of these genes during differentiation, as well as uncertainty regarding disruption of important genomic regions caused by integration. In our study, we performed a non-integrative vector based reprogramming approach using porcine fetal fibroblasts. The resulting four piPSC lines were positive for pluripotency marker and when subjected to in vitro and in vivo differentiation assays, all four lines formed embryoid bodies, capable to differentiate into all three germ layers, and three out of the four cell lines formed teratomas. PCR analysis on genomic and plasmid DNA revealed that the episomal vectors were undetectable in six out of eight subclones derived from one of the piPSC lines (piPSC1) above passage 20. These piPSCs could potentially be ideal cell lines for the generation of porcine in vitro and in vivo models. Furthermore, subsequent analyses of our new transgene independent piPSCs could provide novel insights on the genetic and epigenetic necessities to achieve and maintain piPSCs.

Paving the Way Toward Complex Blood-Brain Barrier Models Using Pluripotent Stem Cells.

A tissue with great need to be modeled in vitro is the blood-brain barrier (BBB). The BBB is a tight barrier that covers all blood vessels in the brain and separates the brain microenvironment from the blood system. It consists of three cell types [neurovascular unit (NVU)] that contribute to the unique tightness and selective permeability of the BBB and has been shown to be disrupted in many diseases and brain disorders, such as vascular dementia, stroke, multiple sclerosis, and Alzheimer's disease. Given the progress that pluripotent stem cells (PSCs) have made in the past two decades, it is now possible to produce many cell types from the BBB and even partially recapitulate this complex tissue in vitro. In this review, we summarize the most recent developments in PSC differentiation and modeling of the BBB. We also suggest how patient-specific human-induced PSCs could be used to model BBB dysfunction in the future. Lastly, we provide perspectives on how to improve production of the BBB in vitro, for example by improving pericyte differentiation protocols and by better modeling the NVU in the dish.

Breaking down pluripotency in the porcine embryo reveals both a premature and reticent stem cell state in the inner cell mass and unique expression profiles of the naive and primed stem cell states.

To date, it has been difficult to establish bona fide porcine embryonic stem cells (pESC) and stable induced pluripotent stem cells. Reasons for this remain unclear, but they may depend on inappropriate culture conditions. This study reports the most insights to date on genes expressed in the pluripotent cells of the porcine embryo, namely the inner cell mass (ICM), the trophectoderm-covered epiblast (EPI), and the embryonic disc epiblast (ED). Specifically, we reveal that the early porcine ICM represents a premature state of pluripotency due to lack of translation of key pluripotent proteins, and the late ICM enters a transient, reticent pluripotent state which lacks expression of most genes associated with pluripotency. We describe a unique expression profile of the porcine EPI, reflecting the naive stem cell state, including expression of OCT4, NANOG, CRIPTO, and SSEA-1; weak expression of NrOB1 and REX1; but very limited expression of genes in classical pathways involved in regulating pluripotency. The porcine ED, reflecting the primed stem cell state, can be characterized by the expression of OCT4, NANOG, SOX2, KLF4, cMYC, REX1, CRIPTO, and KLF2. Further cell culture experiments using inhibitors against FGF, JAK/STAT, BMP, WNT, and NODAL pathways on cell cultures derived from day 5 and 10 embryos reveal the importance of FGF, JAK/STAT, and BMP signaling in maintaining cell proliferation of pESCs in vitro. Together, this article provides new insights into the regulation of pluripotency, revealing unique stem cell states in the different porcine stem cell populations derived from the early developing embryo.

Induced Pluripotent Stem Cells Derived from Alzheimer's Disease Patients: The Promise, the Hope and the Path Ahead.

The future hope of generated induced pluripotent stem cells (iPS cells) from Alzheimer's disease patients is multifold. Firstly, they may help to uncover novel mechanisms of the disease, which could lead to the development of new and unprecedented drugs for patients and secondly, they could also be directly used for screening and testing of potential new compounds for drug discovery. In addition, in the case of familial known mutations, these cells could be targeted by use of advanced gene-editing techniques to correct the mutation and be used for future cell transplantation therapies. This review summarizes the work so far in regards to production and characterization of iPS cell lines from both sporadic and familial Alzheimer's patients and from other iPS cell lines that may help to model the disease. It provides a detailed comparison between published reports and states the present hurdles we face with this new technology. The promise of new gene-editing techniques and accelerated aging models also aim to move this field further by providing better control cell lines for comparisons and potentially better phenotypes, respectively.

Early development of the porcine embryo: the importance of cell signalling in development of pluripotent cell lines.

Understanding the cell signalling events that govern cell renewal in porcine pluripotent cells may help improve culture conditions and allow for establishment of bona fide porcine embryonic stem cells (pESC) and stable porcine induced pluripotent stem cells (piPSC). This review investigates cell signalling in the porcine preimplantation embryo containing either the inner cell mass or epiblast, with particular emphasis on fibroblast growth factor, SMAD, WNT and Janus tyrosine kinases/signal transducers and activators of transcription signalling. It is clear that key differences exist in the cell signalling events that govern pluripotency in this species compared with similar embryonic stages in mouse and human. The fact that bona fide pESC have still not been produced and that piPSC cannot survive in culture following the silencing or downregulation of the reprogramming transgenes suggest that culture conditions are not optimal. Unravelling the factor/s that regulate pluripotency in porcine embryos will pave the way for future establishment of stable pluripotent stem cell lines.

Dynamic changes in epigenetic marks and gene expression during porcine epiblast specification.

Given the difficulties in establishing bona fide porcine embryonic stem cells, we considered it would be interesting to investigate histone modifications, X chromosome inactivation (XCI), deacetylation, DNA methylation, and gene expression around the time of inner cell mass (ICM) and epiblast formation in sexed embryos. We found that the porcine epiblast expressed lower levels of NANOG and C-MYC, of which, we speculate may be one indication for the difficulties in obtaining embryonic stem cells (ESCs) from the porcine embryonic epiblast. Our research revealed distinct expression of lineage-specific-, early gastrulation-, and pluripotency-associated genes between the E10 epiblast and trophectoderm and between sexes. We determined that H3K27me3 was hypermethylated in the E6 embryo and hypomethylated in the E10 epiblast. Interestingly, we also observed exclusive localization of H3K4me3 in the E6 ICM, which may be a key marker for early lineage segregation in the pig. We also observed that the methyltransferases of H3K4me3, H3K27me3 and H3K9me3 and the DNA methyltransferases differed between male and female E10 embryos, and between tissues. We consider that epigenetic mechanisms, which are modified by specific enzymes, may be important for both early lineage segregation events and XCI, and these may further effect the levels of downstream-targeted gene expression in the different sexes.

Regulation of H3K27me3 and H3K4me3 during early porcine embryonic development.

The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co-factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre-implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1- to the 4-cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1-cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4-cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4-cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst.