Biology subjects

Gerbin, K. A.

Publications and source records attributed to Gerbin, K. A..

5 recordsLinked to original sources

bioRxiv2021

A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes

We performed a comprehensive analysis of the transcriptional changes within and across cell populations during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using the single cell RNA-seq combinatorial barcoding method SPLiT-seq, we sequenced >20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. As expected, differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in differentiation population composition and cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.

cell biology↗

bioRxiv2020

Robust integrated intracellular organization of the human iPS cell: where, how much, and how variable?

Despite the intimate link between cell organization and function, the principles underlying intracellular organization and the relation between organization, gene expression and phenotype are not well understood. We address this by creating a benchmark for mean cell organization and the natural range of cell-to-cell variation. This benchmark can be used for comparison to other normal or abnormal cell states. To do this, we developed a reproducible microscope imaging pipeline to generate a high-quality dataset of 3D, high-resolution images of over 200,000 live cells from 25 isogenic human induced pluripotent stem cell (hiPSC) lines from the Allen Cell Collection. Each line contains one fluorescently tagged protein, created via endogenous CRISPR/Cas9 gene editing, representing a key cellular structure or organelle. We used these images to develop a new multi-part and generalizable analysis approach of the locations, amounts, and variation of these 25 cellular structures. Taking an integrated approach, we found that both the extent to which a structures individual location varied ("stereotypy") and the extent to which the structure localized relative to all the other cellular structures ("concordance") were robust to a wide range of cell shape variation, from flatter to taller, smaller to larger, or less to more polarized cells. We also found that these cellular structures varied greatly in how their volumes scaled with cell and nuclear size. These analyses create a data-driven set of quantitative rules for the locations, amounts, and variation of 25 cellular structures within the hiPSC as a normal baseline for cell organization.

cell biology↗

bioRxiv2020

Cell states beyond transcriptomics: integrating structural organization and gene expression in hiPSC-derived cardiomyocytes

We present a quantitative co-analysis of RNA abundance and sarcomere organization in single cells and an integrated framework to predict subcellular organization states from gene expression. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes expressing mEGFP-tagged alpha-actinin-2 to develop quantitative image analysis tools for systematic and automated classification of subcellular organization. This captured a wide range of sarcomeric organization states within cell populations that were previously difficult to quantify. We performed RNA FISH targeting genes identified by single cell RNA sequencing to simultaneously assess the relationship between transcript abundance and structural states in single cells. Co-analysis of gene expression and sarcomeric patterns in the same cells revealed biologically meaningful correlations that could be used to predict organizational states. This study establishes a framework for multi-dimensional analysis of single cells to study the relationships between gene expression and subcellular organization and to develop a more nuanced description of cell states. Graphical AbstractTranscriptional profiling and structural classification was performed on human induced pluripotent stem cell-derived cardiomyocytes to characterize the relationship between transcript abundance and subcellular organization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/081083v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@8f89c4org.highwire.dtl.DTLVardef@19dc813org.highwire.dtl.DTLVardef@1ba7c20org.highwire.dtl.DTLVardef@2b3daa_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Delta-1 functionalized hydrogel promotes hESC-cardiomyocyte graft proliferation and maintains heart function post-injury

Current cell transplantation techniques are hindered by small graft size, requiring high cell doses to achieve therapeutic cardiac remuscularization. Enhancing the proliferation of transplanted human stem cell-derived cardiomyocytes (hESC-CMs) could address this, allowing an otherwise subtherapeutic cell dose to prevent disease progression after myocardial infarction. Here, we designed a hydrogel that activates Notch signaling through 3D presentation of the Notch ligand Delta-1 to use as an injectate for transplanting hESC-CMs into the infarcted rat myocardium. After four weeks, hESC-CM proliferation increased 2-fold and resulted in a 3-fold increase in graft size with the Delta-1 hydrogel compared to controls. To stringently test the effect of Notch-mediated graft expansion on long-term heart function, a normally subtherapeutic dose of hESC-CMs was implanted into the infarcted myocardium and cardiac function was evaluated by echocardiography. Transplantation of the Delta-1 hydrogel + hESC-CMs augmented heart function and was significantly higher at three months compared to controls. Graft size and hESC-CM proliferation were also increased at three months post-implantation. Collectively, these results demonstrate the therapeutic approach of a Delta-1 functionalized hydrogel to reduce the cell dose required to achieve functional benefit after myocardial infarction by enhancing hESC-CM graft size and proliferation.

bioengineering↗

bioRxiv2018

Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells

We describe a multi-step CRISPR/Cas9 gene editing method to create endogenously tagged GFP-fusions of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs), allowing visualization of proteins that are only expressed upon differentiation. To do this, we designed a donor template containing the monomeric EGFP (mEGFP) fusion tag and an mCherry selection cassette delivered in tandem to a target locus via homology directed repair (HDR). mCherry expression was driven by a constitutive promoter and served as a drug-free, excisable selection marker. Following selection, the mCherry cassette was excised with Cas9, creating an mEGFP-fusion with the target gene. We achieved scarless excision by using repetitive sequences to guide microhomology-mediated end joining (MMEJ) and introduce linker sequences between the mEGFP tag and the target gene. Using this strategy, we successfully tagged genes encoding the cardiomyocyte sarcomeric proteins troponin I (TNNI1), alpha-actinin (ACTN2), titin (TTN), myosin light chain 2a (MYL7), and myosin light chain 2v (MYL2) with mEGFP in undifferentiated hiPSCs. This methodology provides a general strategy for scarlessly introducing tags to transcriptionally silent loci in hiPSCs.

cell biology↗