Traumatic brain injury induces TDP-43 mislocalization and neurodegenerative effects in tissue distal to the primary injury site in a non-transgenic mouse.

Traumatic brain injury (TBI) initiates tissue and cellular damage to the brain that is immediately followed by secondary injury sequalae with delayed and continual damage. This secondary damage includes pathological processes that may contribute to chronic neurodegeneration and permanent functional and cognitive deficits. TBI is also associated with an increased risk of developing neurodegenerative diseases such as Alzheimer's disease (AD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) as indicated by shared pathological features. For example, abnormalities in the TAR DNA-binding Protein 43 (TDP-43) that includes cytoplasmic mislocalization, cytosolic aggregation, and an increase in phosphorylation and ubiquitination are seen in up to 50% of FTD cases, up to 70% of AD cases, and is considered a hallmark pathology of ALS occurring in > 97% of cases. Yet the prevalence of TDP-43 pathology post-TBI has yet to be fully characterized. Here, we employed a non-transgenic murine controlled cortical injury model of TBI and observed injury-induced hallmark TDP-43 pathologies in brain and spinal cord tissue distal to the primary injury site and did not include the focally damaged tissue within the primary cortical injury site. Analysis revealed a temporal-dependent and significant increase in neuronal TDP-43 mislocalization in the cortical forebrain rostral to and distant from the primary injury site up to 180 days post injury (DPI). TDP-43 mislocalization was also detected in neurons located in the ventral horns of the cervical spinal cord following a TBI. Moreover, a cortical layer-dependent affect was identified, increasing from superficial to deeper cortical layers over time from 7 DPI up to 180 DPI. Lastly, RNAseq analysis confirmed an injury-induced misregulation of several key biological processes implicated in neurons that increased over time. Collectively, this study demonstrates a connection between a single moderate TBI event and chronic neurodegenerative processes that are not limited to the primary injury site and broadly distributed throughout the cortex and corticospinal tract.

PINE-TREE enables highly efficient genetic modification of human cell lines.

Prime editing technologies enable precise genome editing without the caveats of CRISPR nuclease-based methods. Nonetheless, current approaches to identify and isolate prime-edited cell populations are inefficient. Here, we established a fluorescence-based system, prime-induced nucleotide engineering using a transient reporter for editing enrichment (PINE-TREE), for real-time enrichment of prime-edited cell populations. We demonstrated the broad utility of PINE-TREE for highly efficient introduction of substitutions, insertions, and deletions at various genomic loci. Finally, we employ PINE-TREE to rapidly and efficiently generate clonal isogenic human pluripotent stem cell lines, a cell type recalcitrant to genome editing.

The Multifaceted Role of WNT Signaling in Alzheimer's Disease Onset and Age-Related Progression.

The evolutionary conserved WNT signaling pathway orchestrates numerous complex biological processes during development and is critical to the maintenance of tissue integrity and homeostasis in the adult. As it relates to the central nervous system, WNT signaling plays several roles as it relates to neurogenesis, synaptic formation, memory, and learning. Thus, dysfunction of this pathway is associated with multiple diseases and disorders, including several neurodegenerative disorders. Alzheimer's disease (AD) is characterized by several pathologies, synaptic dysfunction, and cognitive decline. In this review, we will discuss the various epidemiological, clinical, and animal studies that demonstrate a precise link between aberrant WNT signaling and AD-associated pathologies. In turn, we will discuss the manner in which WNT signaling influences multiple molecular, biochemical, and cellular pathways upstream of these end-point pathologies. Finally, we will discuss how merging tools and technologies can be used to generate next generation cellular models to dissect the relationship between WNT signaling and AD.

Modeling long QT syndrome type 2 on-a-chip via in-depth assessment of isogenic gene-edited 3D cardiac tissues.

Long QT syndrome (LQTS) is a cardiovascular disease characterized by QT interval prolongation that can lead to sudden cardiac death. Many mutations with heterogeneous mechanisms have been identified in KCNH2, the gene that encodes for hERG (Kv11.1), which lead to onset of LQTS type 2 (LQTS2). In this work, we developed a LQTS2-diseased tissue-on-a-chip model, using 3D coculture of isogenic stem cell-derived cardiomyocytes (CMs) and cardiac fibroblasts (CFs) within an organotypic microfluidic chip technology. Primarily, we created a hiPSC line with R531W mutation in KCNH2 using CRISPR-Cas9 gene-editing technique and characterized the resultant differentiated CMs and CFs. A deficiency in hERG trafficking was identified in KCNH2-edited hiPSC-CMs, revealing a possible mechanism of R531W mutation in LQTS2 pathophysiology. Following creation of a 3D LQTS2 tissue-on-a-chip, the tissues were extensively characterized, through analysis of calcium handling and response to ß-agonist. Furthermore, attempted phenotypic rescue via pharmacological intervention of LQTS2 on a chip was investigated.

A microcarrier-based protocol for scalable generation and purification of human induced pluripotent stem cell-derived neurons and astrocytes.

Here, we describe a protocol for a microcarrier (MC)-based, large-scale generation and cryopreservation of human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes. We also detail steps to isolate these populations with a high degree of purity. Finally, we describe how to cryopreserve these cell types while maintaining high levels of viability and preserving cellular function post-thaw. For complete details on the use and execution of this protocol, please refer to Brookhouser et al. (2021).

Weighing up the evidence used by direct-to-consumer stem cell businesses.

Hundreds of businesses across the United States offer direct-to-consumer stem-cell-based interventions that have not been approved by the Food and Drug Administration. Here, we characterize the types of evidence used on the websites of 59 stem cell businesses in the Southwest United States to market their services. We identify over a dozen forms of evidence, noting that businesses are less likely to rely on "gold-standard" scientific evidence, like randomized clinical trials, and instead draw substantially on forms of evidence that we identify as being "ambiguous." Ambiguous evidence has some scientific or medical basis, but its interpretation is highly context-dependent. These findings highlight the interpretive responsibility placed on prospective patients. We identify actions for regulators and professional societies to assist with evaluating evidence, but caution that focusing on the (in)validity of particular evidence types is unlikely to eliminate demand for stem-cell-based treatments in this complex marketplace.

Generation of 3X FLAG-tagged human embryonic stem cell (hESC) line to study WNT-induced ß-catenin DNA interactions (HVRDe009-A-2).

In the canonical WNT signaling pathway, active WNT signaling results in the nuclear translocation of ß-catenin where it regulates target gene expression. As a tool to understand these ß-catenin DNA interactions, we used a CRISPR/Cas9 based approach to engineer a human embryonic stem cell line (hESC) harboring a 3X FLAG sequence fused to the C-terminus of ß-catenin. Engineered cells displayed a characteristic hESC morphology, expressed pluripotency-associated markers, retained tri-lineage differentiation potential, and had a normal euploid karyotype. This cell line represents a valuable tool to dissect the transcriptional mechanisms by which WNT signalling regulates pluripotent cell fate.

Apolipoprotein E regulates lipid metabolism and α-synuclein pathology in human iPSC-derived cerebral organoids.

APOE4 is a strong genetic risk factor for Alzheimer's disease and Dementia with Lewy bodies; however, how its expression impacts pathogenic pathways in a human-relevant system is not clear. Here using human iPSC-derived cerebral organoid models, we find that APOE deletion increases α-synuclein (αSyn) accumulation accompanied with synaptic loss, reduction of GBA levels, lipid droplet accumulation and dysregulation of intracellular organelles. These phenotypes are partially rescued by exogenous apoE2 and apoE3, but not apoE4. Lipidomics analysis detects the increased fatty acid utilization and cholesterol ester accumulation in apoE-deficient cerebral organoids. Furthermore, APOE4 cerebral organoids have increased αSyn accumulation compared to those with APOE3. Carrying APOE4 also increases apoE association with Lewy bodies in postmortem brains from patients with Lewy body disease. Our findings reveal the predominant role of apoE in lipid metabolism and αSyn pathology in iPSC-derived cerebral organoids, providing mechanistic insights into how APOE4 drives the risk for synucleinopathies.

Cytosine and adenosine base editing in human pluripotent stem cells using transient reporters for editing enrichment.

Deaminase fused-Cas9 base editing technologies have enabled precise single-nucleotide genomic editing without the need for the introduction of damaging double-stranded breaks and inefficient homology-directed repair. However, current methods to isolate base-edited cell populations are ineffective, especially when utilized with human pluripotent stem cells, a cell type resistant to genome modification. Here, we outline a series of methods that employ transient reporters of editing enrichment (TREE) to facilitate the highly efficient single-base editing of human cells at precise genomic loci. Briefly, these transient reporters of editing enrichment based methods employ a transient episomal fluorescent reporter that allows for the real-time, flow-cytometry-based enrichment of cells that have had single nucleotide changes at precise genomic locations. This protocol details how these approaches can enable the rapid (~3-4 weeks) and efficient (clonal editing efficiencies >80%) generation of biallelic or multiplexed edited isogenic hPSC lines using adenosine and cytosine base editors.

APOE2 mitigates disease-related phenotypes in an isogenic hiPSC-based model of Alzheimer's disease.

Genome-wide association studies (GWAS) have identified polymorphism in the Apolipoprotein E gene (APOE) to be the most prominent risk factor for Alzheimer's disease (AD). Compared to individuals homozygous for the APOE3 variant, individuals with the APOE4 variant have a significantly elevated risk of AD. On the other hand, longitudinal studies have shown that the presence of the APOE2 variant reduces the lifetime risk of developing AD by 40 percent. While there has been significant research that has identified the risk-inducing effects of APOE4, the underlying mechanisms by which APOE2 influences AD onset and progression have not been extensively explored. In this study, we utilize an isogenic human induced pluripotent stem cell (hiPSC)-based system to demonstrate that conversion of APOE3 to APOE2 greatly reduced the production of amyloid-beta (Aß) peptides in hiPSC-derived neural cultures. Mechanistically, analysis of pure populations of neurons and astrocytes derived from these neural cultures revealed that mitigating effects of APOE2 are mediated by cell autonomous and non-autonomous effects. In particular, we demonstrated the reduction in Aß is potentially driven by a mechanism related to non-amyloidogenic processing of amyloid precursor protein (APP), suggesting a gain of the protective function of the APOE2 variant. Together, this study provides insights into the risk-modifying effects associated with the APOE2 allele and establishes a platform to probe the mechanisms by which APOE2 enhances neuroprotection against AD.

Prime Editing Guide RNA Design Automation Using PINE-CONE.

CRISPR-based technologies are paramount in genome engineering and synthetic biology. Prime editing (PE) is a technology capable of installing genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. Prime editing guide RNAs (pegRNAs) simultaneously encode both guide and edit template sequences. They are more design intensive than CRISPR single guide RNAs (sgRNAs). As such, application of PE technology is hindered by the limited throughput of manual pegRNA design. To that end, we designed a software tool, Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE), that enables high-throughput automated design of pegRNAs and prime editing strategies. PINE-CONE translates edit coordinates and sequences into pegRNA designs, accessory guides, and oligonucleotides for facile cloning workflows. To demonstrate PINE-CONE's utility in studying disease-relevant genotypes, we rapidly design a library of pegRNAs targeting Alzheimer's Disease single nucleotide polymorphisms (SNPs). Overall, PINE-CONE will accelerate the application of PEs in synthetic biology and biomedical research.

The Emergence of Model Systems to Investigate the Link Between Traumatic Brain Injury and Alzheimer's Disease.

Numerous epidemiological studies have demonstrated that individuals who have sustained a traumatic brain injury (TBI) have an elevated risk for developing Alzheimer's disease and Alzheimer's-related dementias (AD/ADRD). Despite these connections, the underlying mechanisms by which TBI induces AD-related pathology, neuronal dysfunction, and cognitive decline have yet to be elucidated. In this review, we will discuss the various in vivo and in vitro models that are being employed to provide more definite mechanistic relationships between TBI-induced mechanical injury and AD-related phenotypes. In particular, we will highlight the strengths and weaknesses of each of these model systems as it relates to advancing the understanding of the mechanisms that lead to TBI-induced AD onset and progression as well as providing platforms to evaluate potential therapies. Finally, we will discuss how emerging methods including the use of human induced pluripotent stem cell (hiPSC)-derived cultures and genome engineering technologies can be employed to generate better models of TBI-induced AD.

A Cas9-mediated adenosine transient reporter enables enrichment of ABE-targeted cells.

BACKGROUND: Adenine base editors (ABE) enable single nucleotide modifications without the need for double-stranded DNA breaks (DSBs) induced by conventional CRIPSR/Cas9-based approaches. However, most approaches that employ ABEs require inefficient downstream technologies to identify desired targeted mutations within large populations of manipulated cells. In this study, we developed a fluorescence-based method, named "Cas9-mediated adenosine transient reporter for editing enrichment" (CasMAs-TREE; herein abbreviated XMAS-TREE), to facilitate the real-time identification of base-edited cell populations. RESULTS: To establish a fluorescent-based assay able to detect ABE activity within a cell in real time, we designed a construct encoding a mCherry fluorescent protein followed by a stop codon (TGA) preceding the coding sequence for a green fluorescent protein (GFP), allowing translational readthrough and expression of GFP after A-to-G conversion of the codon to "TGG." At several independent loci, we demonstrate that XMAS-TREE can be used for the highly efficient purification of targeted cells. Moreover, we demonstrate that XMAS-TREE can be employed in the context of multiplexed editing strategies to simultaneous modify several genomic loci. In addition, we employ XMAS-TREE to efficiently edit human pluripotent stem cells (hPSCs), a cell type traditionally resistant to genetic modification. Furthermore, we utilize XMAS-TREE to generate clonal isogenic hPSCs at target sites not editable using well-established reporter of transfection (RoT)-based strategies. CONCLUSION: We established a method to detect adenosine base-editing activity within a cell, which increases the efficiency of editing at multiple genomic locations through an enrichment of edited cells. In the future, XMAS-TREE will greatly accelerate the application of ABEs in biomedical research.

APOE4 exacerbates synapse loss and neurodegeneration in Alzheimer's disease patient iPSC-derived cerebral organoids.

APOE4 is the strongest genetic risk factor associated with late-onset Alzheimer's disease (AD). To address the underlying mechanism, we develop cerebral organoid models using induced pluripotent stem cells (iPSCs) with APOE ε3/ε3 or ε4/ε4 genotype from individuals with either normal cognition or AD dementia. Cerebral organoids from AD patients carrying APOE ε4/ε4 show greater apoptosis and decreased synaptic integrity. While AD patient-derived cerebral organoids have increased levels of Aß and phosphorylated tau compared to healthy subject-derived cerebral organoids, APOE4 exacerbates tau pathology in both healthy subject-derived and AD patient-derived organoids. Transcriptomics analysis by RNA-sequencing reveals that cerebral organoids from AD patients are associated with an enhancement of stress granules and disrupted RNA metabolism. Importantly, isogenic conversion of APOE4 to APOE3 attenuates the APOE4-related phenotypes in cerebral organoids from AD patients. Together, our study using human iPSC-organoids recapitulates APOE4-related phenotypes and suggests APOE4-related degenerative pathways contributing to AD pathogenesis.

Human Autopsy-Derived Scalp Fibroblast Biobanking for Age-Related Neurodegenerative Disease Research.

The Arizona Study of Aging and Neurodegenerative Disorders/Brain and Body Donation Program at Banner Sun Health Research Institute (BSHRI) is a longitudinal clinicopathological study with a current enrollment of more than 900 living subjects for aging and neurodegenerative disease research. Annual clinical assessments are done by cognitive and movement neurologists and neuropsychologists. Brain and body tissues are collected at a median postmortem interval of 3.0 h for neuropathological diagnosis and banking. Since 2018, the program has undertaken banking of scalp fibroblasts derived from neuropathologically characterized donors with Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. Here, we describe the procedure development and cell characteristics from 14 male and 15 female donors (mean ± SD of age: 83.6 ± 12.2). Fibroblasts from explant cultures were banked at passage 3. The results of mRNA analysis showed positive expression of fibroblast activation protein, vimentin, fibronectin, and THY1 cell surface antigen. We also demonstrated that the banked fibroblasts from a postmortem elderly donor were successfully reprogramed to human-induced pluripotent stem cells (hiPSCs). Taken together, we have demonstrated the successful establishment of a human autopsy-derived fibroblast banking program. The cryogenically preserved cells are available for request at the program website of the BSHRI.

Guidelines for establishing a 3-D printing biofabrication laboratory.

Advanced manufacturing and 3D printing are transformative technologies currently undergoing rapid adoption in healthcare, a traditionally non-manufacturing sector. Recent development in this field, largely enabled by merging different disciplines, has led to important clinical applications from anatomical models to regenerative bioscaffolding and devices. Although much research to-date has focussed on materials, designs, processes, and products, little attention has been given to the design and requirements of facilities for enabling clinically relevant biofabrication solutions. These facilities are critical to overcoming the major hurdles to clinical translation, including solving important issues such as reproducibility, quality control, regulations, and commercialization. To improve process uniformity and ensure consistent development and production, large-scale manufacturing of engineered tissues and organs will require standardized facilities, equipment, qualification processes, automation, and information systems. This review presents current and forward-thinking guidelines to help design biofabrication laboratories engaged in engineering model and tissue constructs for therapeutic and non-therapeutic applications.

Engineering anisotropic human stem cell-derived three-dimensional cardiac tissue on-a-chip.

Despite significant efforts in the study of cardiovascular diseases (CVDs), they persist as the leading cause of mortality worldwide. Considerable research into human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has highlighted their immense potential in the development of in vitro human cardiac tissues for broad mechanistic, therapeutic, and patient-specific disease modeling studies in the pursuit of CVD research. However, the relatively immature state of hPSC-CMs remains an obstacle in enhancing clinical relevance ofengineered cardiac tissue models. In this study, we describe development of a microfluidic platform for 3D modeling of cardiac tissues, derived from both rat cells and hPSC-CMs, to better recapitulate the native myocardium through co-culture with interstitial cells (specifically cardiac fibroblasts), biomimetic collagen hydrogel encapsulation, and induction of highly anisotropic tissue architecture. The presented platform is precisely engineered through incorporation of surface topography in the form of staggered microposts to enable long-term culture and maturation of cardiac cells, resulting in formation of physiologically relevant cardiac tissues with anisotropy that mimics native myocardium. After two weeks of culture, hPSC-derived cardiac tissues exhibited well-defined sarcomeric striations, highly synchronous contractions, and upregulation of several maturation genes, including HCN1, KCNQ1, CAV1.2, CAV3.1, PLN, and RYR2. These findings demonstrate the ability of the proposed engineered platform to mature animal- as well as human stem cell-derived cardiac tissues over an extended period of culture, providing a novel microfluidic chip with the capability for cardiac disease modeling and therapeutic testing.

A Defined and Scalable Peptide-Based Platform for the Generation of Human Pluripotent Stem Cell-Derived Astrocytes.

Astrocytes comprise the most abundant cell type in the central nervous system (CNS) and play critical roles in maintaining neural tissue homeostasis. In addition, astrocyte dysfunction and death has been implicated in numerous neurological disorders such as multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). As such, there is much interest in using human pluripotent stem cell (hPSC)-derived astrocytes for drug screening, disease modeling, and regenerative medicine applications. However, current protocols for generation of astrocytes from hPSCs are limited by the use of undefined xenogeneic components and two-dimensional (2D) culture surfaces, which limits their downstream applications where large-quantities of cells generated under defined conditions are required. Here, we report the use of a completely synthetic, peptide-based substrate that allows for the differentiation of highly pure populations of astrocytes from several independent hPSC lines, including those derived from patients with neurodegenerative disease. This substrate, which we demonstrate is compatible with both conventional 2D culture formats and scalable microcarrier (MC)-based technologies, leads to the generation of cells that express high levels of canonical astrocytic markers as well as display properties characteristic of functionally mature cells including production of apolipoprotein E (ApoE), responsiveness to inflammatory stimuli, ability to take up amyloid-ß (Aß), and appearance of robust calcium transients. Finally, we show that these astrocytes can be cryopreserved without any loss of functionality. In the future, we anticipate that these methods will enable the development of bioprocesses for the production of hPSC-derived astrocytes needed for biomedical research and clinical applications.