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1. A Deep Learning Framework for Sickle Cell Disease Microfluidic Biomarker Assays

   https://doi.org/10.1182/blood-2020-141693  

   Praljak, Niksa ; Shipley, Brandon ; Gonzales, Ayesha ; Goreke, Utku ; Iram, Shamreen ; Singh, Gundeep ; Hill, Ailis ; Gurkan, Umut A. ; Hinczewski, Michael ( November 2020 , Blood)

   Introduction: Vaso-occlusive crises (VOCs) are a leading cause of morbidity and early mortality in individuals with sickle cell disease (SCD). These crises are triggered by sickle red blood cell (sRBC) aggregation in blood vessels and are influenced by factors such as enhanced sRBC and white blood cell (WBC) adhesion to inflamed endothelium. Advances in microfluidic biomarker assays (i.e., SCD Biochip systems) have led to clinical studies of blood cell adhesion onto endothelial proteins, including, fibronectin, laminin, P-selectin, ICAM-1, functionalized in microchannels. These microfluidic assays allow mimicking the physiological aspects of human microvasculature and help characterize biomechanical properties of adhered sRBCs under flow. However, analysis of the microfluidic biomarker assay data has so far relied on manual cell counting and exhaustive visual morphological characterization of cells by trained personnel. Integrating deep learning algorithms with microscopic imaging of adhesion protein functionalized microfluidic channels can accelerate and standardize accurate classification of blood cells in microfluidic biomarker assays. Here we present a deep learning approach into a general-purpose analytical tool covering a wide range of conditions: channels functionalized with different proteins (laminin or P-selectin), with varying degrees of adhesion by both sRBCs and WBCs, and in both normoxic and hypoxic environments. Methods: Our neuralmore »networks were trained on a repository of manually labeled SCD Biochip microfluidic biomarker assay whole channel images. Each channel contained adhered cells pertaining to clinical whole blood under constant shear stress of 0.1 Pa, mimicking physiological levels in post-capillary venules. The machine learning (ML) framework consists of two phases: Phase I segments pixels belonging to blood cells adhered to the microfluidic channel surface, while Phase II associates pixel clusters with specific cell types (sRBCs or WBCs). Phase I is implemented through an ensemble of seven generative fully convolutional neural networks, and Phase II is an ensemble of five neural networks based on a Resnet50 backbone. Each pixel cluster is given a probability of belonging to one of three classes: adhered sRBC, adhered WBC, or non-adhered / other. Results and Discussion: We applied our trained ML framework to 107 novel whole channel images not used during training and compared the results against counts from human experts. As seen in Fig. 1A, there was excellent agreement in counts across all protein and cell types investigated: sRBCs adhered to laminin, sRBCs adhered to P-selectin, and WBCs adhered to P-selectin. Not only was the approach able to handle surfaces functionalized with different proteins, but it also performed well for high cell density images (up to 5000 cells per image) in both normoxic and hypoxic conditions (Fig. 1B). The average uncertainty for the ML counts, obtained from accuracy metrics on the test dataset, was 3%. This uncertainty is a significant improvement on the 20% average uncertainty of the human counts, estimated from the variance in repeated manual analyses of the images. Moreover, manual classification of each image may take up to 2 hours, versus about 6 minutes per image for the ML analysis. Thus, ML provides greater consistency in the classification at a fraction of the processing time. To assess which features the network used to distinguish adhered cells, we generated class activation maps (Fig. 1C-E). These heat maps indicate the regions of focus for the algorithm in making each classification decision. Intriguingly, the highlighted features were similar to those used by human experts: the dimple in partially sickled RBCs, the sharp endpoints for highly sickled RBCs, and the uniform curvature of the WBCs. Overall the robust performance of the ML approach in our study sets the stage for generalizing it to other endothelial proteins and experimental conditions, a first step toward a universal microfluidic ML framework targeting blood disorders. Such a framework would not only be able to integrate advanced biophysical characterization into fast, point-of-care diagnostic devices, but also provide a standardized and reliable way of monitoring patients undergoing targeted therapies and curative interventions, including, stem cell and gene-based therapies for SCD. Disclosures Gurkan: Dx Now Inc.: Patents & Royalties; Xatek Inc.: Patents & Royalties; BioChip Labs: Patents & Royalties; Hemex Health, Inc.: Consultancy, Current Employment, Patents & Royalties, Research Funding.« less
2. Stem Cell Based Approaches to Modulate the Matrix Milieu in Vascular Disorders

   https://doi.org/10.3389/fcvm.2022.879977  

   S, Sajeesh ; Dahal, Shataakshi ; Bastola, Suraj ; Dayal, Simran ; Yau, Jimmy ; Ramamurthi, Anand ( June 2022 , Frontiers in Cardiovascular Medicine)

   The extracellular matrix (ECM) represents a complex and dynamic framework for cells, characterized by tissue-specific biophysical, mechanical, and biochemical properties. ECM components in vascular tissues provide structural support to vascular cells and modulate their function through interaction with specific cell-surface receptors. ECM–cell interactions, together with neurotransmitters, cytokines, hormones and mechanical forces imposed by blood flow, modulate the structural organization of the vascular wall. Changes in the ECM microenvironment, as in post-injury degradation or remodeling, lead to both altered tissue function and exacerbation of vascular pathologies. Regeneration and repair of the ECM are thus critical toward reinstating vascular homeostasis. The self-renewal and transdifferentiating potential of stem cells (SCs) into other cell lineages represents a potentially useful approach in regenerative medicine, and SC-based approaches hold great promise in the development of novel therapeutics toward ECM repair. Certain adult SCs, including mesenchymal stem cells (MSCs), possess a broader plasticity and differentiation potential, and thus represent a viable option for SC-based therapeutics. However, there are significant challenges to SC therapies including, but not limited to cell processing and scaleup, quality control, phenotypic integrity in a disease milieu in vivo , and inefficient delivery to the site of tissue injury. SC-derived or -inspired strategies asmore »a putative surrogate for conventional cell therapy are thus gaining momentum. In this article, we review current knowledge on the patho-mechanistic roles of ECM components in common vascular disorders and the prospects of developing adult SC based/inspired therapies to modulate the vascular tissue environment and reinstate vessel homeostasis in these disorders.« less
3. Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy

   Ramamurthy, R ; Zheng, WT ; George, S ; Wan, M ; Zhou, Y ; Lu, B ; Bishop, C ; Atala, A ; Porada, C ; Almeida-Porada, G ( December 2021 , ASH)

   2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed inmore »those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures: No relevant conflicts of interest to declare.« less
4. Development and Utilization of Multifunctional Polymeric Scaffolds for the Regulation of Physical Cellular Microenvironments

   https://doi.org/10.3390/polym13223880  

   Tai, Youyi ; Banerjee, Aihik ; Goodrich, Robyn ; Jin, Lu ; Nam, Jin ( November 2021 , Polymers)

   Polymeric biomaterials exhibit excellent physicochemical characteristics as a scaffold for cell and tissue engineering applications. Chemical modification of the polymers has been the primary mode of functionalization to enhance biocompatibility and regulate cellular behaviors such as cell adhesion, proliferation, differentiation, and maturation. Due to the complexity of the in vivo cellular microenvironments, however, chemical functionalization alone is usually insufficient to develop functionally mature cells/tissues. Therefore, the multifunctional polymeric scaffolds that enable electrical, mechanical, and/or magnetic stimulation to the cells, have gained research interest in the past decade. Such multifunctional scaffolds are often combined with exogenous stimuli to further enhance the tissue and cell behaviors by dynamically controlling the microenvironments of the cells. Significantly improved cell proliferation and differentiation, as well as tissue functionalities, are frequently observed by applying extrinsic physical stimuli on functional polymeric scaffold systems. In this regard, the present paper discusses the current state-of-the-art functionalized polymeric scaffolds, with an emphasis on electrospun fibers, that modulate the physical cell niche to direct cellular behaviors and subsequent functional tissue development. We will also highlight the incorporation of the extrinsic stimuli to augment or activate the functionalized polymeric scaffold system to dynamically stimulate the cells.
5. Pre-Clinical Cell Therapeutic Approaches for Repair of Volumetric Muscle Loss

   https://doi.org/10.3390/bioengineering7030097  

   Shayan, Mahdis ; Huang, Ngan F. ( September 2020 , Bioengineering)

   Extensive damage to skeletal muscle tissue due to volumetric muscle loss (VML) is beyond the inherent regenerative capacity of the body, and results in permanent functional debilitation. Current clinical treatments fail to fully restore native muscle function. Recently, cell-based therapies have emerged as a promising approach to promote skeletal muscle regeneration following injury and/or disease. Stem cell populations, such as muscle stem cells, mesenchymal stem cells and induced pluripotent stem cells (iPSCs), have shown a promising capacity for muscle differentiation. Support cells, such as endothelial cells, nerve cells or immune cells, play a pivotal role in providing paracrine signaling cues for myogenesis, along with modulating the processes of inflammation, angiogenesis and innervation. The efficacy of cell therapies relies on the provision of instructive microenvironmental cues and appropriate intercellular interactions. This review describes the recent developments of cell-based therapies for the treatment of VML, with a focus on preclinical testing and future trends in the field.