MitoCellPhe reveals mitochondrial morphologies in single fibroblasts and clustered stem cells.

Mitochondria are dynamic organelles that differ significantly in their morphologies across cell types, reflecting specific cellular needs and stages in development. Despite the wide biological significance in disease and in health, delineating mitochondrial morphologies in complex systems remains challenging. Here, we present the Mitochondrial Cellular Phenotype (MitoCellPhe) tool developed for quantifying mitochondrial morphologies and demonstrate its utility in delineating differences in mitochondrial morphologies in a human fibroblast and human induced pluripotent stem cell (hiPSC) line. MitoCellPhe generates 24 parameters, allowing for a comprehensive analysis of mitochondrial structures and importantly allows for quantification to be performed on mitochondria in images containing single cells or clusters of cells. With this tool, we were able to validate previous findings that show networks of mitochondria in healthy fibroblast cell lines and a more fragmented morphology in hiPSCs. Using images generated from control and diseased fibroblasts and hiPSCs, we also demonstrate the efficacy of the toolset in delineating differences in morphologies between healthy and the diseased state in both stem cell (hiPSC) and differentiated fibroblast cells. Our results demonstrate that MitoCellPhe enables high-throughput, sensitive, detailed, and quantitative mitochondrial morphological assessment and thus enables better biological insights into mitochondrial dynamics in health and disease.

Generation and Characterization of Human Mesenchymal Stem Cell-Derived Smooth Muscle Cells.

Cardiovascular diseases are the leading cause of death worldwide. A completely autologous treatment can be achieved by using elastogenic mesenchymal stem cell (MSC)-derived smooth muscle cells (SMC) at the affected tissue site of vascular diseases such as abdominal aortic aneurysms (AAA). Thus, our work focused on evaluating the efficacy of (a) the combination of various growth factors, (b) different time periods and (c) different MSC lines to determine the treatment combination that generated SMCs that exhibited the greatest elastogenicity among the tested groups using Western blotting and flow cytometry. Additionally, total RNA sequencing was used to confirm that post-differentiation cells were upregulating SMC-specific gene markers. Results indicated that MSCs cultured for four days in PDGF + TGFß1 (PT)-infused differentiation medium showed significant increases in SMC markers and decreases in MSC markers compared to MSCs cultured without differentiation factors. RNA Seq analysis confirmed the presence of vascular smooth muscle formation in MSCs differentiated in PT medium over a seven-day period. Overall, our results indicated that origin, growth factor treatment and culture period played a major role in influencing MSC differentiation to SMCs.

Micellear Gold Nanoparticles as Delivery Vehicles for Dual Tyrosine Kinase Inhibitor ZD6474 for Metastatic Breast Cancer Treatment.

The therapeutic index of poorly water-soluble drugs is often hampered due to poor pharmacokinetics, reduced blood retention, and lack of effective drug concentrations in the tumor region. In order to overcome these issues, drugs are often delivered by use of delivery vehicles to provide an enhanced therapeutic index. Gold nanoparticles synthesized in micellar networks of amphiphilic block copolymer (AuNM) provide an efficient nanocarrier for tissue- and site-specific drug delivery owing to their low cytotoxicity and immunogenicity. AuNM is formed by exploiting the properties of both inorganic Au material and an amphiphilic polymer of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG). We further functionalized AuNM with the FDA-approved dual tyrosine kinase inhibitor ZD6474 and studied the physicochemical properties of the conjugate ZD6474-AuNM. Both AuNM and ZD6474-AuNM, with a diameter of ∼70 nm, were very stable at physiological pH. Conversely, at an acidic pH of 5.2, a slow sustained-release profile of ZD6474 was evident from AuNM, which could provide a method of facilitating release of the drug in an acidic tumor environment. In vitro, in triple-negative breast cancer cells, ZD6474-AuNM inhibited tumor cell proliferation, migration, and invasion and induced apoptosis. There was no detectable lysis of red blood cells observed when they were treated with AuNM and ZD6474-AuNM, confirming hemocompatibility. To reinforce the possibility of AuNM serving as a delivery vehicle, AuNM was conjugated with the IR680 dye for tracking, and this conjugate was systemically delivered in female nude mice bearing MDA-MB-231 human breast cancer xenografts. Fluorescence signal was retained in the tumor region in a temporal manner as compared to other organs, indicating passive retention of AuNM in the tumor locale. Moreover, delivery of ZD6474-AuNM in nude mice bearing MDA-MB-231 xenografts led to decreased tumor size as compared to the control group. The promising safety, targeting, and therapeutic results of systemic delivery of ZD6474 by AuNM provide an attractive alternative method for treating patients with metastatic breast cancer.

Lung cancer stem cells, p53 mutations and MDM2.

Over the past few decades, advances in cancer research have enabled us to understand the different mechanisms that contribute to the aberrant proliferation of normal cells into abnormal cells that result in tumors. In the pursuit to find cures, researchers have primarily focused on various molecular level changes that are unique to cancerous cells. In humans, about 50 % or more cancers have a mutated tumor suppressor p53 gene thereby resulting in accumulation of p53 protein and losing its function to activate the target genes that regulate cell cycle and apoptosis. Extensive research conducted in murine cancer models with activated p53, loss of p53, or p53 missense mutations have facilitated researchers to understand the role of this key protein. Despite the identification of numerous triggers that causes lung cancer specific cure still remain elusive. One of the primary reasons attributed to this is due to the fact that the tumor tissue is heterogeneous and contains numerous sub-populations of cells. Studies have shown that a specific sub-population of cells termed as cancer stem cells (CSCs) drive the recurrence of cancer in response to standard chemotherapy. These CSCs are mutated cells with core properties similar to those of adult stem cells. They reside in a microenvironment within the tumor tissue that supports their growth and make them less susceptible to drug treatment. These cells possess properties of symmetric self-renewal and migration thus driving tumor formation and metastasis. Therefore, research specifically targeting these cells has gained prominence towards developing new therapeutic agents against cancer. This chapter focuses on lung cancer stem cells, p53 mutations noted in these cells, and importance of MDM2 interactions. Further, research approaches for better understanding of molecular mechanisms that drive CSC function and developing appropriate therapies are discussed.

The challenges and prospects of smooth muscle tissue engineering.

Many vascular disorders arise as a result of dysfunctional smooth muscle cells. Tissue engineering strategies have evolved as key approaches to generate functional vascular smooth muscle cells for use in cell-based precision and personalized regenerative medicine approaches. This article highlights some of the challenges that exist in the field and presents some of the prospects for translating research advancements into therapeutic modalities. The article emphasizes the need for better developing synergetic intracellular and extracellular cues in the processes to generate functional vascular smooth muscle cells from different stem cell sources for use in tissue engineering strategies.

This paper explores the potential of engineering smooth muscle tissues to treat vascular diseases, focusing on challenges like sourcing the right cells and creating supportive environments for cell growth. It highlights advances in materials that mimic the body's conditions and the use of 3D fabrication methods for creating complex structures. Additionally, it discusses the significance of mitochondrial function in blood vessel muscle cells. The research emphasizes interdisciplinary efforts and personalized treatments as key to developing effective therapies. The goal is to engineer lab-grown tissues that can repair or replace damaged blood vessels, offering hope for addressing major health challenges associated with vascular diseases.

Immersive virtual reality-based learning as a supplement for biomedical engineering labs: challenges faced and lessons learned.

Introduction: Immersive virtual reality (VR) based laboratory demonstrations have been gaining traction in STEM education as they can provide virtual hands-on experience. VR can also facilitate experiential and visual learning and enhanced retention. However, several optimizations of the implementation, in-depth analyses of advantages and trade-offs of the technology, and assessment of receptivity of modern techniques in STEM education are required to ensure better utilization of VR-based labs. Methods: In this study, we developed VR-based demonstrations for a biomolecular engineering laboratory and assessed their effectiveness using surveys containing free responses and 5-point Likert scale-based questions. Insta360 Pro2 camera and Meta Quest 2 headsets were used in combination with an in-person lab. A cohort of 53 students watched the experimental demonstration on VR headsets in the lab after a brief lab overview in person and then performed the experiments in the lab. Results: Only 28.29% of students reported experiencing some form of discomfort after using the advanced VR equipment as opposed to 63.63% of students from the previous cohort. About 40% of the students reported that VR eliminated or reduced auditory and visual distractions from the environment, the length of the videos was appropriate, and they received enough information to understand the tasks. Discussion: The traditional lab method was found to be more suitable for explaining background information and lab concepts while the VR was found to be suitable for demonstrating lab procedures and tasks. Analyzing open-ended questions revealed several factors and recommendations to overcome the potential challenges and pitfalls of integrating VR with traditional modes of learning. This study provides key insights to help optimize the implementation of immersive VR to effectively supplement in-person learning experiences.

Differentiation and Engineering of Human Stem Cells for Smooth Muscle Generation.

Cardiovascular diseases are responsible for 31% of global deaths and are considered the main cause of death and disability worldwide. Stem cells from various sources have become attractive options for a range of cell-based therapies for smooth muscle tissue regeneration. However, for efficient myogenic differentiation, the stem cell characteristics, cell culture conditions, and their respective microenvironments need to be carefully assessed. This review covers the various approaches involved in the regeneration of vascular smooth muscles by conditioning human stem cells. This article delves into the different sources of stem cells used in the generation of myogenic tissues, the role of soluble growth factors, use of scaffolding techniques, biomolecular cues, relevance of mechanical stimulation, and key transcription factors involved, aimed at inducing myogenic differentiation. Impact statement The review article's main goal is to discuss the recent advances in the field of smooth muscle tissue regeneration. We look at various cell sources, growth factors, scaffolds, mechanical stimuli, and factors involved in smooth muscle formation. These stem cell-based approaches for vascular muscle formation will provide various options for cell-based therapies with long-term beneficial effects on patients.

Early introduction of 3D modeling modules promotes the development of simulation skills in downstream biomedical engineering curricula.

BACKGROUND: Recent advancements in additive manufacturing have made 3D design a desirable skill in combating the historically slow development of biomedical products. Due to the broad applicability of additive manufacturing to biomedical engineering, 3D design and 3D printing are attractive educational tools for biomedical engineering students. However, due to the multidisciplinary nature of biomedical engineering, finding a suitable spot in the curriculum to teach students basic and application-based skills for 3D manufacturing is difficult. Furthermore, prior training in fundamental 3D design skills may be needed to support the use of application-based supplementary content. RESULTS: We designed a SolidWorks Simulations toolkit to complement a sophomore (2nd-year)-level Biomechanics course and distributed this assignment to students with and without prior training in 3D design delivered in an introductory biomedical engineering course. Using short videos, example-based problem solving, and step-by-step tutorials, students completed this as an extra-credit assignment and completed a survey gauging student opinion on SolidWorks and 3D design, confidence in each target skill, and the effectiveness of assignment delivery. The compilation of survey responses suggests that the assignment effectively increased positive responses in student opinion on interest in and likeliness to use SolidWorks in both groups. However, confidence in the target assignment skills was higher in the trained group and fewer problems occurred in operating SolidWorks for trained students. Further, analyzing the distribution of student grades with respect to survey responses suggests that responses had no relationship with initial class grade. CONCLUSION: These data collectively indicate that prior training provided to the students had a positive impact on the effectiveness of the assignment although increases in student opinion on the utility of 3D design were observed in both trained and untrained students. Our work has generated and identified a useful educational supplement to enrich existing biomedical engineering course materials with practical skills.

Clinical utility of mesenchymal stem/stromal cells in regenerative medicine and cellular therapy.

Mesenchymal stem/stromal cells (MSCs) have been carefully examined to have tremendous potential in regenerative medicine. With their immunomodulatory and regenerative properties, MSCs have numerous applications within the clinical sector. MSCs have the properties of multilineage differentiation, paracrine signaling, and can be isolated from various tissues, which makes them a key candidate for applications in numerous organ systems. To accentuate the importance of MSC therapy for a range of clinical indications, this review highlights MSC-specific studies on the musculoskeletal, nervous, cardiovascular, and immune systems where most trials are reported. Furthermore, an updated list of the different types of MSCs used in clinical trials, as well as the key characteristics of each type of MSCs are included. Many of the studies mentioned revolve around the properties of MSC, such as exosome usage and MSC co-cultures with other cell types. It is worth noting that MSC clinical usage is not limited to these four systems, and MSCs continue to be tested to repair, regenerate, or modulate other diseased or injured organ systems. This review provides an updated compilation of MSCs in clinical trials that paves the way for improvement in the field of MSC therapy.

Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential.

BACKGROUND: Human mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype. METHODS: hiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer. RESULTS: Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential. CONCLUSION: Overall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites.

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.

Identification of metabolic changes in genetically unstable stem cells by using model analysis of gene expression.

Stem-cell research seeks to address many different questions related to fundamental stem-cell function with the ultimate goal of being able to control and utilize stem cells for a broad range of therapeutic needs. While a large amount of work is focused on discovering and controlling differentiation mechanisms in stem cells, an equally interesting and important area of work is to understand the basics of stem-cell propagation and self-renewal. With high-throughput genomics and transcriptomic information on hand, it is becoming possible to address some of the detailed mechanistic processes occurring in stem cells, though interpretation of these data is often difficult. In this work, stem cells with genetic abnormalities were compared to genetically normal stem cells using gene-expression array data integrated with a large-scale metabolic model to help interpret changes in metabolism resulting in the identification of several metabolic pathways that were different in the normal and abnormal cells.

A comparative evaluation of layer-by-layer assembly techniques for surface modification of microcarriers used in human mesenchymal stromal cell manufacturing.

The demand for large quantities of highly potent human mesenchymal stromal cells (hMSCs) is growing given their therapeutic potential. To meet high production needs, suspension-based cell cultures using microcarriers are commonly used. Microcarriers are commonly made of or coated with extracellular matrix proteins or charged compounds to promote cell adhesion and proliferation. In this work, a simple method (draining filter) to perform layer by layer (LbL) assembly on microcarriers to create multilayers of heparin and collagen and further demonstrate that these multilayers have a positive effect on hMSC viability after 48 h of culture was demonstrated. The draining filter method is evaluated against two other methods found in literature-centrifugation and fluidized bed, showing that the draining filter method can perform the surface modification with greater efficiency and with less materials and steps needed in the coating process.

Stem cell-based strategies for skeletal muscle tissue engineering.

Skeletal muscle tissue engineering has been a key area of focus over the years and has been of interest for developing regenerative strategies for injured or degenerative skeletal muscle tissue. Stem cells have gained increased attention as sources for developing skeletal muscle tissue for subsequent studies or potential treatments. Focus has been placed on understanding the molecular pathways that govern skeletal muscle formation in development to advance differentiation of stem cells towards skeletal muscle fates in vitro. Use of growth factors and transcription factors have long been the method for guiding skeletal muscle differentiation in vitro. However, further research in small molecule induced differentiation offers a xeno-free option that could result from use of animal derived factors.

Incorporating immersive learning into biomedical engineering laboratories using virtual reality.

BACKGROUND: The Covid-19 pandemic caused a sudden shift towards remote learning, moving classes to online formats. Not exempt from this switch, laboratory courses traditionally taught in-person were also moved to remote methods, costing students the opportunity to learn these skills hands-on. In order for instructors to provide course materials effectively and engagingly, non-traditional methods should be explored. Virtual reality (VR) has become more accessible in recent years. VR simulations have been used for many years as educational tools in high-risk settings such as flight or medical simulations. Immersive VR videos implemented in a remote laboratory course could provide the students with an engaging and suitable learning experience. To test the effectiveness of VR videos as a tool for remote education, VR videos of the laboratory component of a Biomolecular Engineering course were provided to students. A survey was distributed for students to self-report their experience with the videos. The survey contained quantitative and qualitative ratings of VR as an educational tool. RESULTS: The survey showed that students (~ 89% strongly agree or agree) believed the videos provided the opportunity to work at their own pace and were an appropriate length. While ~ 74% of students said that the videos provided enough information to understand the tasks, a small percentage felt that the videos improved their retention (~ 16%) and understanding (~ 9%) of the course material. About 28% of the students responded positively when asked about how VR videos improved their engagement with the material. ~ 30% reported confidence in applying the skills learned in the videos in the future and ~ 43% believe the VR videos were an acceptable alternative to in-person labs. Two-thirds of students reported feeling some form of discomfort while viewing the VR videos and 54% reported not using the headset for the videos and using the 3D video feature instead. CONCLUSIONS: As many students reported the videos containing appropriate information, the content of the videos was not an issue. A combination of improved camera quality with motion stability, more comfortable headsets, and a reduction in editing issues could greatly improve the quality and effectiveness of VR videos.

Immunomodulatory functions of human mesenchymal stromal cells are enhanced when cultured on HEP/COL multilayers supplemented with interferon-gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for cell therapies due to their immunosuppressive capacity that can be enhanced in the presence of interferon-gamma (IFN-γ). In this study, multilayers of heparin (HEP) and collagen (COL) (HEP/COL) were used as a bioactive surface to enhance the immunomodulatory activity of hMSCs using soluble IFN-γ. Multilayers were formed, via layer-by-layer assembly, varying the final layer between COL and HEP and supplemented with IFN-γ in the culture medium. We evaluated the viability, adhesion, real-time growth, differentiation, and immunomodulatory activity of hMSCs on (HEP/COL) multilayers. HMSCs viability, adhesion, and growth were superior when cultured on (HEP/COL) multilayers compared to tissue culture plastic. We also confirmed that hMSCs osteogenic and adipogenic differentiation remained unaffected when cultured in (HEP/COL) multilayers in the presence of IFN-γ. We measured the immunomodulatory activity of hMSCs by measuring the level of indoleamine 2,3-dioxygenase (IDO) expression. IDO expression was higher on (HEP/COL) multilayers treated with IFN-γ. Lastly, we evaluated the suppression of peripheral blood mononuclear cell (PBMC) proliferation when co-cultured with hMSCs on (HEP/COL) multilayers with IFN-γ. hMSCs cultured in (HEP/COL) multilayers in the presence of soluble IFN-γ have a greater capacity to suppress PBMC proliferation. Altogether, (HEP/COL) multilayers with IFN-γ in culture medium provides a potent means of enhancing and sustaining immunomodulatory activity to control hMSCs immunomodulation.

Cell-Permeable Succinate Increases Mitochondrial Membrane Potential and Glycolysis in Leigh Syndrome Patient Fibroblasts.

Mitochondrial disorders represent a large group of severe genetic disorders mainly impacting organ systems with high energy requirements. Leigh syndrome (LS) is a classic example of a mitochondrial disorder resulting from pathogenic mutations that disrupt oxidative phosphorylation capacities. Currently, evidence-based therapy directed towards treating LS is sparse. Recently, the cell-permeant substrates responsible for regulating the electron transport chain have gained attention as therapeutic agents for mitochondrial diseases. We explored the therapeutic effects of introducing tricarboxylic acid cycle (TCA) intermediate substrate, succinate, as a cell-permeable prodrug NV118, to alleviate some of the mitochondrial dysfunction in LS. The results suggest that a 24-hour treatment with prodrug NV118 elicited an upregulation of glycolysis and mitochondrial membrane potential while inhibiting intracellular reactive oxygen species in LS cells. The results from this study suggest an important role for TCA intermediates for treating mitochondrial dysfunction in LS. We show, here, that NV118 could serve as a therapeutic agent for LS resulting from mutations in mtDNA in complex I and complex V dysfunctions.

Quantitative analysis of mitochondrial morphologies in human induced pluripotent stem cells for Leigh syndrome.

Mitochondria are dynamic organelles with wide range of morphologies contributing to regulating different signaling pathways and several cellular functions. Leigh syndrome (LS) is a classic pediatric mitochondrial disorder characterized by complex and variable clinical pathologies, and primarily affects the nervous system during early development. It is important to understand the differences between mitochondrial morphologies in healthy and diseased states so that focused therapies can target the disease during its early stages. In this study, we performed a comprehensive analysis of mitochondrial dynamics in five patient-derived human induced pluripotent stem cells (hiPSCs) containing different mutations associated with LS. Our results suggest that subtle alterations in mitochondrial morphologies are specific to the mtDNA variant. Three out of the five LS-hiPSCs exhibited characteristics consistent with fused mitochondria. To our knowledge, this is the first comprehensive study that quantifies mitochondrial dynamics in hiPSCs specific to mitochondrial disorders. In addition, we observed an overall decrease in mitochondrial membrane potential in all five LS-hiPSCs. A more thorough analysis of the correlations between mitochondrial dynamics, membrane potential dysfunction caused by mutations in the mtDNA in hiPSCs and differentiated derivatives will aid in identifying unique morphological signatures of various mitochondrial disorders during early stages of embryonic development.

ARHGDIA Confers Selective Advantage to Dissociated Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) have generated significant interest in the scientific community based on their potential applications in regenerative medicine. However, numerous research groups have reported a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. Work from our laboratory and others has demonstrated that amplification of chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during clinical use. In this study, we focus on trisomy 17 and a list of candidate genes with increased gene expression to hypothesize that overexpressing 17q25 located ARHGDIA will confer selective advantage to hPSCs. HPSC lines overexpressing ARHGDIA exhibited culture dominance in co-cultures of overexpression lines with nonoverexpression lines. Furthermore, during low-density seeding, we demonstrate increased clonality of our ARHGDIA lines against matched controls. A striking observation is that we could reduce this selective advantage by varying the hPSC culture conditions with the addition of ROCK inhibitor (ROCKi). This work is unique in (1) demonstrating a novel gene that confers selective advantage to hPSCs when overexpressed and may help explain a common trisomy dominance, (2) providing a selection model for studying culture conditions that reduce the appearance of genomically altered hPSCs, and (3) aiding in elucidation of a mechanism that may act as a molecular switch during culture adaptation.

Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system.

We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols.