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.

Cervical spine degenerative disease is an independent risk factor for increased revision rate following total knee arthroplasty.

INTRODUCTION: The relationship between cervical degenerative pathology and total knee arthroplasty (TKA) revision rates is not well understood. The aim of the study was to determine whether cervical spine degenerative diseases have a role in complications following TKA within 2 years. METHODS: Data were collected from the Humana insurance database using the PearlDiver Patient Records Database from 2007-2017. Patients who had a primary TKA were identified using Current Procedural Terminology (CPT) code 27,447, and patients with degenerative cervical disease were identified using CPT and International Classification of Diseases (ICD) codes. Data on patients' demographics, comorbidities and postoperative complications were recorded and analyzed with univariate and multivariate analysis with significance set at p < 0.05. A Kaplan-Meier analysis was conducted to estimate the 1- and 2-year rates of survival free from revision. RESULTS: A total of 81,873 patients were included in this study. Following multivariate analysis, cervical spine degenerative disease patients were at increased risk of all-cause revision surgery following 1 year (OR: 1.342 95% CI: 1.149-1.569; p < 0.001) and 2 year (OR: 1.338; 95% CI: 1.184-1.512; p < 0.001). At 2 years, patients with cervical spine degenerative disease had a survival rate of 97.7%, while the survival rate was 99.2% among the non-cervical degenerative cohort. CONCLUSIONS: Based on these results, patients with cervical spine degenerative pathology should be counseled that their spinal pathology may impair outcomes following TKA.

3D Printed scaffolds with hierarchical biomimetic structure for osteochondral regeneration.

Osteochondral defects resulting from trauma and/or pathologic disorders are critical clinical problems. The current approaches still do not yield satisfactory due to insufficient donor sources and potential immunological rejection of implanted tissues. 3D printing technology has shown great promise for fabricating customizable, biomimetic tissue matrices. The purpose of the present study is to investigate 3D printed scaffolds with biomimetic, biphasic structure for osteochondral regeneration. For this purpose, nano-hydroxyapatite and transforming growth factor beta 1 nanoparticles were synthesized and distributed separately into the lower and upper layers of the biphasic scaffold, which was fabricated using 3D stereolithography printer. Our results showed that this scaffold design successfully promoted osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells, as well as enhanced gene expression associated with both osteogenesis and chondrogenesis alike. The finding demonstrated that 3D printed osteochondral scaffolds with biomimetic, biphasic structure are excellent candidates for osteochondral repair and regeneration.

3D bioprinting mesenchymal stem cell-laden construct with core-shell nanospheres for cartilage tissue engineering.

Cartilage tissue is prone to degradation and has little capacity for self-healing due to its avascularity. Tissue engineering, which provides artificial scaffolds to repair injured tissues, is a novel and promising strategy for cartilage repair. 3D bioprinting offers even greater potential for repairing degenerative tissue by simultaneously integrating living cells, biomaterials, and biological cues to provide a customized scaffold. With regard to cell selection, mesenchymal stem cells (MSCs) hold great capacity for differentiating into a variety of cell types, including chondrocytes, and could therefore be utilized as a cartilage cell source in 3D bioprinting. In the present study, we utilize a tabletop stereolithography-based 3D bioprinter for a novel cell-laden cartilage tissue construct fabrication. Printable resin is composed of 10% gelatin methacrylate (GelMA) base, various concentrations of polyethylene glycol diacrylate (PEGDA), biocompatible photoinitiator, and transforming growth factor beta 1 (TGF-ß1) embedded nanospheres fabricated via a core-shell electrospraying technique. We find that the addition of PEGDA into GelMA hydrogel greatly improves the printing resolution. Compressive testing shows that modulus of the bioprinted scaffolds proportionally increases with the concentrations of PEGDA, while swelling ratio decreases with the increase of PEGDA concentration. Confocal microscopy images illustrate that the cells and nanospheres are evenly distributed throughout the entire bioprinted construct. Cells grown on 5%/10% (PEGDA/GelMA) hydrogel present the highest cell viability and proliferation rate. The TGF-ß1 embedded in nanospheres can keep a sustained release up to 21 d and improve chondrogenic differentiation of encapsulated MSCs. The cell-laden bioprinted cartilage constructs with TGF-ß1-containing nanospheres is a promising strategy for cartilage regeneration.

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.

Integrating three-dimensional printing and nanotechnology for musculoskeletal regeneration.

The field of tissue engineering is advancing steadily, partly due to advancements in rapid prototyping technology. Even with increasing focus, successful complex tissue regeneration of vascularized bone, cartilage and the osteochondral interface remains largely illusive. This review examines current three-dimensional printing techniques and their application towards bone, cartilage and osteochondral regeneration. The importance of, and benefit to, nanomaterial integration is also highlighted with recent published examples. Early-stage successes and challenges of recent studies are discussed, with an outlook to future research in the related areas.

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.

High antimicrobial effectiveness with low hemolytic and cytotoxic activity for PEG/quaternary copolyoxetanes.

The alkyl chain length of quaternary ammonium/PEG copolyoxetanes has been varied to discern effects on solution antimicrobial efficacy, hemolytic activity and cytotoxicity. Monomers 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) were used to prepare precursor P[(BBOx)(ME2Ox)-50:50-4 kDa] copolyoxetane via cationic ring opening polymerization. The 1:1 copolymer composition and Mn (4 kDa) were confirmed by (1)H NMR spectroscopy. After C-Br substitution by a series of tertiary amines, ionic liquid Cx-50 copolyoxetanes were obtained, where 50 is the mole percent of quaternary repeat units and "x" is quaternary alkyl chain length (2, 6, 8, 10, 12, 14, or 16 carbons). Modulated differential scanning calorimetry (MDSC) studies showed Tgs between -40 and -60 °C and melting endotherms for C14-50 and C16-50. Minimum inhibitory concentrations (MIC) were determined for Escherichia coli , Staphylococcus aureus , and Pseudomonas aeruginosa . A systematic dependence of MIC on alkyl chain length was found. The most effective antimicrobials were in the C6-50 to C12-50 range. C8-50 had better overall performance with MICs of 4 µg/mL, E. coli ; 2 µg/mL, S. aureus ; and 24 µg/mL, P. aeruginosa . At 5 × MIC, C8-50 effected >99% kill in 1 h against S. aureus , E. coli , and P. aeruginosa challenges of 10(8) cfu/mL; log reductions (1 h) were 7, 3, and 5, respectively. To provide additional insight into polycation interactions with bacterial membranes, a geometric model based on the dimensions of E. coli is described that provides an estimate of the maximum number of polycations that can chemisorb. Chain dimensions were estimated for polycation C8-50 with a molecular weight of 5 kDa. Considering the approximations for polycation chemisorption (PCC), it is surprising that a calculation based on geometric considerations gives a C8-50 concentration within a factor of 2 of the MIC, 4.0 (±1.2) µg/mL for E. coli . Cx-50 copolyoxetane cytotoxicity was low for human red blood cells, human dermal fibroblasts (HDF), and human foreskin fibroblasts (HFF). Selectivities for bacterial kill over cell lysis were among the highest ever reported for polycations indicating good prospects for biocompatibility.

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.

Radiographic characterization of prevertebral soft tissue shadow in the cervicothoracic region of normal adults.

BACKGROUND: Many studies have found measurement of prevertebral soft tissue shadow (PVSTS) on a lateral cervical radiograph to be a useful indicator of cervical spine injury. The purpose of this study is to define, measure, and establish a normative set of values for radiographic width of the PVSTS in the cervicothoracic region of the spine (C7-T4), using swimmer's view in subjects with no trauma to the region. MATERIALS AND METHODS: Radiographic PVSTS widths were measured at each vertebral level from C7 to T4 on 131 patients who had "normal" radiographic examination (mean age 31.5 y, range 18-58 y). Intra-observer repeatability was assessed on a random subset of 24 subjects. The range, mean, and standard error of these measurements were calculated and documented. Stepwise forward regression analysis was conducted on PVSTS data and those normalized with respect to the C7 vertebral body width (rPVSTS) to study the influences of age, sex, disk level, and tracheal curve shape. RESULTS: Regression analysis showed that, in order of influence, the vertebral level, sex, and age were three significant factors that affected PVSTS, whereas tracheal curve shape was not significant. Similar results were obtained using normalized rPVSTS data, with the exception that the influence of sex was not significant in this instance. CONCLUSIONS: This study provides a reliable normative database of PVSTS in the North American population, and shows that measurement of prevertebral soft tissue shadow on a swimmer's view radiograph can be used as a valuable screening tool in the evaluation of cervicothoracic spine injury.

Biomechanical comparison of supplemental posterior fixations for two-level anterior lumbar interbody fusion.

Posterior instrumentations have been used to supplement anterior lumbar interbody fusion with cages. Biomechanical studies on single-level anterior lumbar interbody fusion show that stand-alone cages supplemented with posterior translaminar facet or transfacet screw fixation exhibit comparable stability to those supplemented with pedicle screw/rod fixation, while stability of multilevel anterior lumbar interbody fusion remains mostly unknown. The objectives of this study are to compare the stabilization of three supplemental posterior fixations to two-level anterior lumbar interbody fusion, including translaminar facet fixation, transfacet screw fixation, and pedicle screw/rod fixation. Flexibility tests were conducted on fresh-frozen calf spines with moment up to 8.5 N m in flexion, extension, lateral bending, and axial rotation. Each specimen was tested at three stages: intact, anterior lumbar interbody fusion using Polyetheretherketone (PEEK) interbody cage at L3-L4 and L4-L5, and the same anterior lumbar interbody fusion plus one of the three supplemental posterior fixations. The addition of the supplemental posterior fixation increased stiffness at the fusion levels significantly in flexion (9.9 times), extension (5.4 times), and lateral bending (4.1 times). The pedicle screw/rod and translaminar screw fixations provide approximately 40% higher stiffness than the transfacet screw in lateral bending. The pedicle screw/rod fixation also displayed a trend of superior fixation in extension. Supplemental posterior fixation significantly improved stability of two-level anterior lumbar interbody fusion when compared to the stand-alone cages. Pedicle screw/rod system is still the "gold standard" in providing supplemental stability. However, both translaminar facet screws and transfacet screws are good alternatives to provide adequate fixation.

Single-level transforaminal interbody fusion for traumatic lumbosacral fracture-dislocation: a case report.

L5S1 fracture-dislocations are rare three-column injuries. The infrequency of this injury has led to a lack of a universally accepted treatment strategy. Transforaminal lumbar interbody fusion (TLIF) has been shown to be an effective approach for interbody fusion in degenerative indications, but has not been previously reported in the operative management of traumatic lumbosacral dislocation. The authors report a case of traumatic L5S1 fracture-dislocation in a 30-year-old male, presenting with a right-sided L5 neurologic deficit, following a street sweeper accident. Imaging revealed an L5S1 fracture-dislocation with fracture of the S1 body. Open reduction with TLIF and L5S1 posterolateral instrumented fusion was carried out within 24 hours of injury. Excellent reduction was obtained, and maintained at long-term follow-up, with complete resolution of pain and neurologic deficit. In this patient, L5S1 fracture-dislocation was treated successfully, with an excellent outcome, with a single level TLIF and instrumented posterolateral fusion at L5S1.

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.

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.

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.