Ibrahim J Domian’s research while affiliated with Harvard Medical School and other places

Dynamic profiling of changes in gene expression in response to stressors in specific microenvironments without requiring cellular destruction remains challenging. Current methodologies that seek to interrogate gene expression at a molecular level require sampling of cellular transcriptome and therefore lysis of the cell, preventing serial analysis of cellular transcriptome. To address this area of unmet need, we have recently developed a technology allowing transcriptomic analysis over time without cellular destruction. Our method, TRACE-seq (TRanscriptomic Analysis Captured in Extracellular vesicles using sequencing), is characterized by a cell-type specific transgene expression. It provides data on the transcriptome inside extracellular vesicles that provides an accurate representation of stress-responsive cellular transcriptomic changes. Thus, the transcriptome of cells expressing TRACE can be followed over time without destroying the source cell, which is a powerful tool for many fields of fundamental and translational biology research.

Changes in gene expression due to cell heterogeneity and response to stressors in specific microenvironments remains poorly understood. Current methodologies that seek to interrogate gene expression at a molecular level require sampling of cellular transcriptome and therefore lysis of the cell preventing serial analysis of cellular transcriptome. To address this area of unmet need, we have recently developed a new technology allowing transcriptomic analysis over time without cellular destruction. Our novel method, TRACE-seq (TRanscriptomic Analysis Captured in Extracellular vesicles using sequencing), is characterized by a cell-type specific transgene expression. It provides data on a representative part of the cell transcriptome inside extracellular vesicles. Thus, the transcriptome of cells expressing TRACE can be followed over time in a nondestructive manner, which is a powerful tool for many fields of fundamental and translational biology research.

Background & Aim In vitro differentiation of human induced pluripotent stem cells into cardiomyocytes (hiPSC-CMs) is a crucial process to enable their application in cell therapy and drug discovery. Despite the remarkable efforts over the last decade towards the optimization of cardiac differentiation protocols, there are some technological challenges remaining, including the low scalability and differentiation yields. Additionally, generated hiPSC-CM are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype, structure and function. Our work aims to devise novel bioinspired strategies to improve the generation and functionality of hiPSC-CM. Noteworthy, we integrated structural and functional analyses of hPSC-CM with powerful “omics” technologies as to support bioprocess understanding and cell product characterization. Methods, Results & Conclusion We relied on the aggregation of hiPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. Whole-transcriptome and ¹³C-metabolic flux analyses demonstrated that a three-dimensional (3D) and agitated-based culture environment improves CM purity, functionality and metabolic maturation. We also assessed if alteration of culture medium composition to recapitulate in vivo substrate usage during heart development improved further hiPSC-CM maturation in vitro. By shifting hiPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium, we promoted their fast maturation into adult-like CMs with higher oxidative metabolism, higher myofibril density and alignment as well as improved calcium handling and contractility. This study demonstrated for the first time that metabolic shifts during differentiation/maturation of hiPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The bioinspired metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. We are now applying the generated hiPSC-CM for the development of physiologically-relevant cardiac tissues. By modulating key environmental factors namely, electrical and physical stimuli, co-culture with other hiPSC-cardiac derivatives, biomaterials as well as components of the ECM, we were able to improve further hiPSC-CM maturation features and provide novel insight associated with heart regenerative mechanisms.

Defining the pathways that control cardiac development facilitates understanding the pathogenesis of congenital heart disease. Herein, we identify enrichment of a Cullin5 Ub ligase key subunit, Asb2, in myocardial progenitors and differentiated cardiomyocytes. Using two conditional murine knockouts: Nkx+/Cre.Asb2fl/fl and AHF-Cre.Asb2fl/fl and tissue clarifying technique, we reveal Asb2 requirement for embryonic survival and complete heart looping. Deletion of Asb2 results in upregulation of its target FilaminA (Flna) and concurrent Flna deletion partially rescues embryonic lethality. Conditional AHF-Cre.Asb2 knockouts harboring one Flna allele have double outlet right ventricle (DORV) which is rescued by bi-allelic Flna excision. Transcriptomic and immunofluorescence analyses identify Tgfβ/Smad as downstream targets of Asb2/Flna. Finally, using CRISPR/Cas9 genome editing, we demonstrate Asb2 requirement for human cardiomyocyte differentiation suggesting a conserved mechanism between mice and humans. Collectively, our study provides deeper mechanistic understanding of the UPS role in cardiac development and suggests a previously unidentified murine model for DORV.

A fundamental goal in the biological sciences is to determine how individual cells with varied gene expression profiles and diverse functional characteristics contribute to development, physiology, and disease. Here, we report a novel strategy to assess gene expression and cell physiology in single living cells. Our approach utilizes fluorescently-labeled mRNA-specific anti-sense RNA probes and dsRNA-binding protein to identify the expression of specific genes in real-time at single-cell resolution via FRET. We use this technology to identify distinct myocardial subpopulations expressing the structural proteins myosin heavy chain α and myosin light chain 2a in real-time during early differentiation of human pluripotent stem cells. We combine this live-cell gene expression analysis with detailed physiologic phenotyping to capture the functional evolution of these early myocardial subpopulations during lineage specification and diversification. This live-cell mRNA imaging approach will have wide ranging application wherever heterogeneity plays an important biological role.

Here we propose a bio‐MEMS device designed to evaluate contractile force and conduction velocity of cell sheets in response to mechanical and electrical stimulation of the cell source as it grows to form a cellular sheet. Moreover, the design allows for the incorporation of patient‐specific data and cell sources. An optimized device would allow cell sheets to be cultured, characterized, and conditioned to be compatible with a specific patient's cardiac environment in vitro, before implantation. This design draws upon existing methods in the literature but makes an important advance by combining the mechanical and electrical stimulation into a single system for optimized cell sheet growth. The device has been designed to achieve cellular alignment, electrical stimulation, mechanical stimulation, conduction velocity readout, contraction force readout, and eventually cell sheet release. The platform is a set of comb electrical contacts consisting of three‐dimensional walls made of polydimethylsiloxane and coated with electrically conductive metals on the tops of the walls. Not only do the walls serve as a method for stimulating cells that are attached to the top, but their geometry is tailored such that they are flexible enough to be bent by the cells and used to measure force. The platform can be stretched via a linear actuator setup, allowing for simultaneous electrical and mechanical stimulation that can be derived from patient‐specific clinical data.

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.

Ccbe1 knockdown does not seem to affect normal Brachyury expression pattern between days 0 and 6. qPCR analysis at days 0, 2, 4 and 6 of the general mesoderm marker brachyury (BraT). Analysis was performed in two individual Ccbe1 KD ESC clones (Clone 1 and Clone 2) and relative expression is compared to day 0 of each cell type. Mean ± SEM of three biological replicates in technical qPCR triplicates. (TIF)

Primers for qPCR gene expression analysis. (DOCX)

Post-cardiac surgical sternal and epicardial adhesions increase the risk and complexity of cardiac re-operative surgeries, which represent a significant challenge for patients with the congenital cardiac disease. Bioresorbable membranes can serve as barriers to prevent postoperative adhesions. Herein, we fabricated a bioresorbable gelatin/polycaprolactone (GT/PCL) composite membrane via electrospinning. The membrane was characterized in terms of morphology, mechanical properties, and biocompatibility. We then evaluated its efficacy as a physical barrier to prevent cardiac operative adhesions in a rabbit model. Our results showed that the membrane had a nanofibrous structure and was sturdy enough to be handled for the surgical procedures. In vitro studies with rabbit cardiac fibroblasts demonstrated that the membrane was biocompatible and inhibited cell infiltration. Further application of the membrane in a rabbit cardiac adhesion model revealed that the membrane was resorbed gradually and effectively resisted the sternal and epicardial adhesions. Interestingly, six months after the operation, the GT/PCL membrane was completely resorbed with simultaneous ingrowth of host cells to form a natural barrier. Collectively, these results indicated that the GT/PCL membrane might be a suitable barrier to prevent sternal and epicardial adhesions and might be utilized as a novel pericardial substitute for cardiac surgery. Statement of Significance: Electrospinning is a versatile method to prepare nanofibrous membranes for tissue engineering and regenerative medicine applications. However, with the micro-/nano-scale structure and high porosity, the electrospun membrane might be an excellent candidate as a barrier to prevent postoperative adhesion. Here we prepared an electropun GT/PCL nanofibrous membrane and applied it as a barrier to prevent sternal and epicardial adhesions. Our results showed that the membrane had sufficient mechanical strength, good biocompatibility, and effectively resisted the sternal and epicardial adhesions. What's more, the membrane was bioresorbable and allowed simultaneous ingrowth of host cells to form a natural barrier. We believe that the current will inspire more research on nanomaterials to prevent postoperative adhesion applications.