Collaborative Research Projects (CRPs)

A major part of the CESCG mission is to establish a Collaborative Research Program (CRP) to support the genomics research needs of stem cell investigators in California. Through the CRP funding of individual collaborative research projects, the CESCG will provide expertise and resources for the development and application of new and innovative genomic and epigenomic approaches for human stem cell biology and regenerative medicine. The objective is to combine stem cell resources with CESCG genomic and bioinformatics approaches to accelerate fundamental understanding of human biology and disease mechanisms, enhance cell and tissue production and advance personalized cellular therapeutics.

Comprehensive CRPs (Call 1)

C2 - Geschwind (UCLA): Transcriptional networks in Autism Spectrum Disorder

Advances in genetics and genomics over the last decade have led to the identification of specific genetic variants that account for approximately 20% of the risk for autism spectrum disorder (ASD). Many of these variants represent rare, large effect size mutations.  The Geschwind lab has demonstrated a convergent pattern of disrupted gene expression in post mortem ASD brain, but it is not known how multiple distinct mutations lead to this convergent molecular pathology. To address this question, the lab will perform large-scale genomic and phenotypic characterization of induced pluripotent stem cell (iPSC)-based models of ASD, representing patients with distinct ASD risk mutations, patients with idiopathic ASD and healthy controls. Neurons will be generated from iPSC using an innovative 3D neural differentiation system that generates a laminated human cortex, including synaptically connected neurons and astrocytes. These in vitro models will be phenotyped for morphological and physiological abnormalities using automated assays and entire transcriptional networks will be analyzed during in vitro development. In addition, whole genome sequence will be obtained and together these data will be used to identify potential causal factors and key regulatory drivers of the disease. This will provide new mechanistic insight in ASD pathophysiology and provide an invaluable and unprecedented resource for the field. Furthermore, this project leverages iPSC lines generated as part of the CIRM hiPSC Initiative and capitalizes on tools being developed by the Ideker lab as part of the broader CESCG toolbox.

C3 - Kriegstein (UCSF): A single cell resource of human neural gene expression for improving cell replacement therapies and disease models

Cortical progenitor cells give rise to the diverse populations of cortical neurons. Many protocols have been developed to generate cortical neurons from pluripotent stem cells, including several recent protocols that use aggregate culture methods to differentiate human pluripotent stem cells into cerebral organoids. Cerebral organoids consist of heterogeneous populations of progenitors and neurons but the extent to which these in vitro-derived cell types resemble their endogenous counterparts remains unknown. To address this issue, the Kriegstein lab will use single cell RNA profiling at multiple stages of cerebral organoid differentiation and compare these in vitro gene expression profiles with data they are generating for primary human cortical tissues. These comparisons will provide objective measures of the efficiency and accuracy of various differentiation protocols. The ultimate goal of this analysis is to improve differentiation protocols, a necessary effort in order to realize the potential of stem cells for cell replacement therapy as well as for disease modeling.

Regular CRPs (Call 1)

R4 - Sanford (UCSC): Comparative genomic analysis of alternative splicing and translational control in neurodifferentiation

Alternative pre-mRNA splicing contributes to the regulation of gene expression and protein diversity. Many human diseases, including Amyotrophic Lateral Sclerosis, Frontotemporal Lobar Degeneration and cancer, are caused or exacerbated by aberrant RNA processing. The goals of this study are to investigate RNA processing during neuronal differentiation of human pluripotent stem cells, using innovative genomics approaches. The knowledge gained is not only critical for understanding how splicing is regulated to control differentiation but also for discovering how genetic variants such as inherited disease mutations disrupt gene expression and function.

Regular CRPs (Call 2)

R7 - Corn (UC Berkeley): Using NGS to develop a safe and effective Cas9-based therapy for Sickle Cell disease

Sickle cell disease (SCD) is a devastating genetic disorder that affects ~100,000 primarily African American individuals in the USA, including 5,100 in California. In SCD, a Glu to Val point mutation in the ß-globin gene renders the resultant sickle hemoglobin prone to polymerize and damage the red blood cell. We have used CRISPR-Cas9 genome editing to develop methods to correct the sickle allele in hematopoietic stem cells (HSCs) and, together with SCD experts at Children’s Hospital Oakland, are in the process of developing proof-of-concept for a clinical trial to cure SCD via transplantation of gene-corrected autologous HSCs from patients. Our goal in this CESCG CRP is to establish the efficacy and safety of sickle correction in HSCs via targeted and unbiased sequencing. Together with the CESCG we will use next-generation sequencing to determine the extent of allele conversion in edited HSCs both in vitro and after in vivo engraftment in a mouse model. To establish the safety of editing, we will use three sequencing-based approaches. First, we will use custom amplicon-based resequencing to quantify undesired editing events at related globin genes and at sites computationally predicted as potential off-targets based on sequence similarity. Second, we will use established cancer resequencing panels to uncover low-frequency off-target events at genes known to be involved in tumorigenesis, with a focus on annotated tumor suppressors. Third, we will use unbiased capture and sequencing methods, such as the recently described GUIDE-Seq method, to uncover off-target events in the context of the entire human genome. This approach will be critical in providing key data to move editing SCD allele towards the clinic and will also provide an important precedent for establishing efficacy and safety metrics for therapeutic gene editing in HSCs.

R9 - Loring (Scripps): Sequence analysis of iPSC cells and derivatives for QC of an autologous cell therapy for Parkinson's disease

We are performing preclinical studies for an autologous cell therapy for Parkinson's disease. We have generated iPSCs from 10 Parkinson's patients for our initial cohort, based on criteria established from earlier studies using fetal tissue. Our clinical partner, Dr. Melissa Houser, is Director of the Movement Disorders Clinic at the Scripps Clinic in La Jolla. Our preliminary data include optimized, robust, and reproducible methods for differentiating different lines of patient-specific iPSCs into dopamine neuron precursors, which have shown efficacy in animal models. These are the cell preparations that we plan to transplant to patients to restore their motor control. We also cite our recently published data on genomic stability of human pluripotent stem cells, which pinpoint genomic aberrations that occur when these cells are cultured for very long (>2 years) periods. We include another of our studies, in press in Nature Communications, in which we performed comprehensive analysis of human iPSCs produced by three different reprogramming methods, using whole genome sequencing and de novo genome mapping methods to show that it is unlikely that reprogramming itself will introduce mutations that compromise the safety of iPSCs for therapy. For this grant application, we request funds to perform whole genome and RNA sequencing as quality control measures to assure that the cells that are used for transplantation have no deleterious mutations and are the correct cell type. The mRNAseq studies will expand on our earlier work, funded by CIRM, in which we developed a genomic diagnostic test to determine whether human cells are pluripotent. This gene expression-based diagnostic test, called PluriTest®, is now the most popular assay for pluripotency, recommended by the NIH and used more than 12,000 times since the website ( became active. The genome sequencing studies will improve upon our earlier assessments of genomic stability based on SNP genotyping. These studies will allow us to provide the most rigorous predictive assessments of cell therapy safety and efficacy for clinical stem cell applications, and help to set the standards for future clinical studies using human stem cell-derived products.

R10 - Weissman (Stanford): Charting a single-cell expression atlas of human mesoderm development and hematopoiesis

The goal of this program is to address a fundamentally unsolved issue in human development and stem-cell differentiation: how does human mesoderm become diversified into an array of therapeutically-relevant tissues, including bone, cartilage, skeletal muscle, heart, kidneys and blood? A comprehensive understanding of how these tissues all differentiate from a common embryonic mesodermal source might enable us to artificially generate these diverse lineages from human embryonic stem cells (hESCs) for regenerative medicine. Human mesoderm development remains a terra incognita, because it unfolds during human embryonic weeks 2-4, when it is ethically impossible to retrieve fetuses for developmental analyses. Because human mesoderm ontogeny has never been systematically described, the identity of key developmental intermediates and the order of lineage transitions remain unclear. A clear “lineage tree” of mesoderm development would expand developmental and cell biology and enhance the therapeutic potential of regenerative medicine. Hence our goal is to chart a comprehensive map of human mesoderm development, including the specifics of blood development (hematopoiesis), and to systematically reconstruct its component lineage intermediates and their progenitor-progeny relationships.

R11 - Yeo (UCSD): Large-scale assessment of RNA localization and single-cell alternative splicing in neuronal stem cell models of disease

In recent years, the importance of post-transcriptional gene regulation (PTGS) underlying neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer’s and Parkinson’s disease has increased tremendously with the growing number of RNA binding proteins (RBPs) found mutated in patients. Specifically, despite the hundreds of mutations found within these RBPs in patients with ALS, we still do not understand (1) how and why they cause motor neuron degeneration while leaving other cell-types in the central nervous system such as glial cells relatively unscathed, and (2) what aspects of PTGS these mutations affect in these cells. Thus this represents an unmet medical need. In order to study whether these disease-associated mutations result in cell-type specific PTGS, we propose to utilize induced pluripotent stem cells (iPSCs) from ALS patients harboring disease-causing mutations and generate mature neurons, a highly relevant cell-type in ALS. RBPs interact with specific sequences or structural features within transcribed RNAs to affect PTGS. In this proposal we will test specific hypotheses that these mutant RBPs affect cell-type specific alternative splicing and sub-cellular mis-localization of target mRNAs, both highly relevant to known normal functions of these RBPs. We will focus on abundantly expressed RBPs (TDP-43, FUS/TLS, hnRNP A2/B1 and Matrin3) that have been implicated in ALS. We already have generated patient-specific iPSC lines with TDP-43, FUS/TLS and hnRNP A2/B1 mutations. We will use highly optimized protocols to differentiate iPSCs to neurons. For the first time, we will identify mutant-dependent sub-cellular mis-localization of alternative isoform mRNAs in neurons as a novel hypothesis for disease pathology in ALS. To address whether these mutations cause cell-type specific aberrations in alternative splicing, we will utilize single-cell RNA-seq analysis to measure alternative splicing in the neuron culture system, which contains the requisite heterogeneous mixture of glial cells to maintain a healthy neuron differentiation and physiology. For the first time, we will identify mutant-dependent cell-type specific alternative splicing as a hypothesis for disease pathogenesis. These stem-cell based mRNA signatures are a critical resource that we will compare to post-mortem patient material to identify potential therapeutic targets.