Blitz Talks

The largest organ in the body, skeletal muscle, has remarkable regenerative capacity. Interstitial cells regulate the balance among fibrous, adipose, bone and muscle tissue upon injury. During embryonic development, we have shown that Hox11 genes are expressed in the muscle interstitium and that loss of Hox11 genes disrupts muscle patterning. However, whether Hox11 genes continue to be expressed and function in adult skeletal muscle has not been investigated. Here, we show that Hox11 genes continue to be expressed and overlap with other interstitial cells in adult muscle interstitium. Notably, Hoxa11-expressing muscle cells can differentiate into adipocytes in vitro. The Wellik lab has previously published loss of Hox11 function in bone stromal cells show increased adipogenic potential in vitro. Therefore, we are currently investigating whether the loss of Hox11 function impairs skeletal muscle regeneration in vitro and in vivo and leads to adipogenic accumulation in response to injury.

Cardiovascular diseases encompass a broad range of diseases which account for the highest rates of morbidity and mortality in the United States and in the world. Differentiating iPSCs to cardiomyocytes (CMs) can be used for downstream applications to study these diseases. However, current methods produce inconsistent levels of differentiation with high batch-to-batch variability and low levels of maturation in iPSC-derived CMs (iPSC-CMs). This can, in part, be attributed to the widespread use of Matrigel, a mouse basement-membrane-producing-tumor extract, as a platform for iPSC-CM culture. Our work proposes to optimize the iPSC-CM microenvironment by manipulating hydrogel biochemical and mechanical properties to enhance the levels of differentiation and maturation of these cells. A series of screens were developed to evaluate the effect of different adhesion ligands and hydrogel stiffnesses on iPSC-CM differentiation. Results indicate that hydrogel conditions consistently improved levels of iPSC-CM differentiation compared to Matrigel. Hydrogel conditions showing highest improvement included physiological stiffnesses and cyclic RGD ligands.

Brain mural cells (pericytes and vascular smooth muscle cells), regulate brain vascular development and function. Forebrain mural cells are neural crest (NC)-derived, but molecular signals controlling the differentiation process are poorly understood. Further, existing primary and human pluripotent stem cell (hPSC)-derived in vitro models of brain mural cells have markedly reduced expression of key genes, including NOTCH3, compared to cells in vivo. We asked whether activation of Notch3 in hPSC-derived NC could direct differentiation of mural cells with improved phenotype. We transduced NC with Notch3 intracellular domain (N3ICD)-GFP or GFP. Compared to GFP, N3ICD-GFP-transduced cells exhibited upregulated expression of mural cell transcripts PDGFRB, RGS5, FOXS1, TBX2, and HEYL, and increased protein-level expression of PDGFRβ and both full-length and cleaved Notch3. Thus, our data suggest that activation of Notch3 signaling is sufficient to direct differentiation of NC to brain mural cells, and that multiple mural cell transcription factors are downstream of Notch3. We also establish an improved, serum-free differentiation protocol for hPSC-derived brain mural cells.

Hypertrophic cardiomyopathy (HCM) is a highly prevalent cardiovascular disease yet the early molecular mechanisms leading to HCM remain largely unknown due to insufficient model systems. 3-dimensional engineered cardiac tissue (ECT) constructs made from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have emerged as appealing models due to their closer representation of the structural and functional complexity of the heart. We generated hiPSC-ECT constructs from patients harboring a R663H mutation in myosin heavy chain to mimic the early stage HCM. Sequential assessment of functional properties and top-down mass spectrometry-based proteomics were performed on the same hiPSC-ECTs from both HCM and control lines. Our results indicate that the kinetics, twitch force magnitude, and sarcomere proteoform landscape were significantly altered in the HCM models compared to the control. Global proteomics data also suggests that the extracellular matrix contributes significantly to the HCM phenotype, offering new insights into this "disease of the sarcomere". We hope our integrated methodology empowers further studies for cardiac disease modeling and drug discovery.

Numerous protocols exist for differentiation of human pluripotent stem cells (hPSCs) to cardiomyocytes (CMs). Although these methods have improved in efficiency over the past decade, they remain highly variable in their resultant purities. This substantial heterogeneity of hPSC-CM product outcomes points to poorly-understood, highly sensitive, and uncontrolled variables present within the overall process. Herein, we have undertaken a multi-omic discovery approach to identify key temporal differences in cell attributes between high- and low-purity hPSC-CM differentiations to provide systems-level insights into underlying mechanisms which drive these divergent outcomes. In addition to gaining fundamental insights into the underlying biology of the differentiation process, we are extending our analyses to 1) identify putative quality attributes for continuous process monitoring, 2) enhance process, and 3) establish novel feedback/feedforward control schemes for biomanufacturing processes. Current single-omic analyses are beginning to elucidate interesting temporal trends in differential markers/pathways. Ongoing multi-omic integration seeks to further our insights at the systems level.

Demyelinating diseases such as multiple sclerosis (MS), are debilitating diseases where the degenerative and regenerative pathways are not fully understood. Adult neural stem cells (aNSCs) lining the lateral ventricles in the mammalian brain have remyelinating potential. Following demyelination, these aNSCs can migrate differentiate into myelinating OLs, but the pathways governing this process are largely unknown. We analyzed the transcriptome of aNSCs expressing or lacking Gli1 during demeylination, we identified Gpnmb as significantly altered in aNSCs with enhanced remyelination. Global loss of Gpnmb resulted in enhanced generation of OLs from Gli1 aNSCs following demyelination. Loss of Gpnmb downregulated the expression of TGFβR2, while in vitro TGFβ1 treatment increased Gpnmb suggesting Gpnmb acts downstream or in tandem with TGFβ1. Overexpressing Gpnmb in aNSCs lead to upregulation of TGFβR2 along with inhibition of mature OL generation in vitro. Genetic loss of TGFβR2 specifically in Gli1 aNSCs enhanced OL generation during remyelination. Thus, TGFβ1 induces Gpnmb expression, which upregulates TGFβR2 to amplify this inhibitory pathway in aNSCs and prevent OL regeneration.