Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer

Embryonic and Adult Stem Cells Explored through Microfluidics and Biological Manipulation

Part I - A Microfluidic Method for the Selection of Undifferentiated Human Embryonic Stem Cells and in Situ AnalysisConventional cell-sorting methods such as FACS or MACS can suffer from certain shortcomings such as lengthy sample preparation time, cell modification through antibody labeling, and exposure to high shear forces or metallic microparticles. In light of these drawbacks, we have recently developed a novel, label-free, microfluidic platform that can not only sort cells with minimal sample preparation but also enable analysis of cells in situ. In contrast to MACS or FACS, cells sorted by our method have very high viability (~90%). In this part of my thesis, I first describe existing antibody-functionalized microfluidic devices for cell sorting as well those designed for human embryonic stem cell (hESC) sorting. I then demonstrate the utility of our platform to sort undifferentiated human embryonic stem cells (hESCs) from a heterogeneous population, achieving ~60% average purity of the cells expressing a marker of interest. I also discuss future strategies to improve sorting efficiency. Overall, our platform technology could be applied to other cell types beyond hESCs and to a variety of heterogeneous cell populations.Part II - Attenuation of TGF-β Signaling via Incorporation of a Dominant-negative TGF-β Type II Receptor Promotes Improved Muscle Regeneration in Murine Skeletal MyoblastsSkeletal muscle stem cells known as satellite cells are responsible for muscle regeneration. Upon muscle injury or exercise, quiescent satellite cells become activated, proliferate as myogenic precursors, differentiate into myoblasts, and ultimately fuse into new, multinucleated myofibers. Unfortunately, this paradigm breaks down with aging and multiple factors contribute to a build-up of scar tissue instead of new muscle. Among these negative contributors are some members of the TGF-β family of signaling molecules. After an introduction to muscle regeneration and satellite cells, the adult skeletal muscle stem cells, I will discuss how biomaterials can help improve muscle regeneration and recent advances in combating TGF-β-induced impairment in muscle repair. I will then discuss the work I have done in improving skeletal muscle repair by attenuating the effects of TGF-β signaling via incorporation of a dominant-negative TGF-β type II receptor

Embryonic and Adult Stem Cells Explored through Microfluidics and Biological Manipulation

Part I - A Microfluidic Method for the Selection of Undifferentiated Human Embryonic Stem Cells and in Situ AnalysisConventional cell-sorting methods such as FACS or MACS can suffer from certain shortcomings such as lengthy sample preparation time, cell modification through antibody labeling, and exposure to high shear forces or metallic microparticles. In light of these drawbacks, we have recently developed a novel, label-free, microfluidic platform that can not only sort cells with minimal sample preparation but also enable analysis of cells in situ. In contrast to MACS or FACS, cells sorted by our method have very high viability (~90%). In this part of my thesis, I first describe existing antibody-functionalized microfluidic devices for cell sorting as well those designed for human embryonic stem cell (hESC) sorting. I then demonstrate the utility of our platform to sort undifferentiated human embryonic stem cells (hESCs) from a heterogeneous population, achieving ~60% average purity of the cells expressing a marker of interest. I also discuss future strategies to improve sorting efficiency. Overall, our platform technology could be applied to other cell types beyond hESCs and to a variety of heterogeneous cell populations.Part II - Attenuation of TGF-β Signaling via Incorporation of a Dominant-negative TGF-β Type II Receptor Promotes Improved Muscle Regeneration in Murine Skeletal MyoblastsSkeletal muscle stem cells known as satellite cells are responsible for muscle regeneration. Upon muscle injury or exercise, quiescent satellite cells become activated, proliferate as myogenic precursors, differentiate into myoblasts, and ultimately fuse into new, multinucleated myofibers. Unfortunately, this paradigm breaks down with aging and multiple factors contribute to a build-up of scar tissue instead of new muscle. Among these negative contributors are some members of the TGF-β family of signaling molecules. After an introduction to muscle regeneration and satellite cells, the adult skeletal muscle stem cells, I will discuss how biomaterials can help improve muscle regeneration and recent advances in combating TGF-β-induced impairment in muscle repair. I will then discuss the work I have done in improving skeletal muscle repair by attenuating the effects of TGF-β signaling via incorporation of a dominant-negative TGF-β type II receptor

Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer

Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer

Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a powerful tool for human disease modeling and therapeutic testing. However, their use remains limited by their immaturity and heterogeneity. To characterize the source of this heterogeneity, we applied complementary single-cell RNA-seq and bulk RNA-seq technologies over time during hiPSC cardiac differentiation and in the adult heart. Using integrated transcriptomic and splicing analysis, more than half a dozen distinct single-cell populations were observed, several of which were coincident at a single time-point, day 30 of differentiation. To dissect the role of distinct cardiac transcriptional regulators associated with each cell population, we systematically tested the effect of a gain or loss of three transcription factors (NR2F2, TBX5, and HEY2), using CRISPR genome editing and ChIP-seq, in conjunction with patch clamp, calcium imaging, and CyTOF analysis. These targets, data, and integrative genomics analysis methods provide a powerful platform for understanding in vitro cellular heterogeneity

Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis

Human induced pluripotent stem cell derived cardiomyocytes are a powerful model for cardiogenesis and disease in vitro. Here the authors comprehensively map cardiac differentiation using multiple modalities, including single-cell RNA seq and CyTOF, in cells with a gain  or loss of function in key cardiac transcription factors