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Each heartbeat that pumps blood throughout the body is initiated by an electrical impulse generated in the sinoatrial node (SAN). However, a number of disease conditions can hamper the ability of the SAN′s pacemaker cells to generate consistent action potentials and maintain an orderly conduction path, leading to arrhythmias. For symptomatic patients, current treatments rely on implantation of an electronic pacing device. However, complications inherent to the indwelling hardware give pause to categorical use of device therapy for a subset of populations, including pediatric patients or those with temporary pacing needs. Cellular-based biological pacemakers, derived in vitro or in situ, could function as a therapeutic alternative to current electronic pacemakers. Understanding how biological pacemakers measure up to the SAN would facilitate defining and demonstrating its advantages over current treatments. In this review, we discuss recent approaches to creating biological pacemakers and delineate design criteria to guide future progress based on insights from basic biology of the SAN. We emphasize the need for long-term efficacy in vivo via maintenance of relevant proteins, source-sink balance, a niche reflective of the native SAN microenvironment, and chronotropic competence. With a focus on such criteria, combined with delivery methods tailored for disease indications, clinical implementation will be attainable.

Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.

On March 1 and 2, 2018, the National Institutes of Health 2018 Progenitor Cell Translational Consortium (PCTC) and Cardiovascular Bioengineering Symposium (CVBE) was held at the University of Alabama at Birmingham. Convergence of life sciences and engineering to advance the understanding and treatment of heart failure was the theme of the meeting. Over 150 attendees were present for more than 40 scientists presenting their latest works on engineering human functional myocardium for disease modeling, drug development, and heart failure research. The scientists, engineers and physicians in the field of cardiovascular sciences, met and discussed on the most recent advances in their works and propose future strategies in overcoming the major roadblocks of cardiovascular bioengineering and therapy. Particular emphasis was given for manipulation and using of stem/progenitor cells, biomaterials, and methods to provide molecular, chemical and mechanical cues to cells in order to influence their identity and fate in vitro and in vivo. Collectively, these works are profoundly impacting and progressing toward deciphering the mechanisms and developing novel treatments for left ventricular dysfunction of failing hearts. Here we present some important perspectives that emerged from this meeting.

Rationale: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes—a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. Objective: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. Methods and Results: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell–laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. Conclusions: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

Recessive dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in collagen-type VII gene critical for the dermoepidermal junction (DEJ) formation. Neither tissues of animal models nor currently available in vitro models are amenable to the quantitative assessment of mechanical adhesion between dermal and epidermal layers. Here, we created a 3D in vitro DEJ model using extracellular matrix (ECM) proteins of the DEJ anchored to a poly(ethylene glycol)-based slab (termed ECM composites) and seeded with human keratinocytes and dermal fibroblasts. Keratinocytes and fibroblasts of healthy individuals were well maintained in the ECM composite and showed the expression of collagen type VII over a 2-week period. The ECM composites with healthy keratinocytes and fibroblasts exhibited yield stress associated with the separation of the model DEJ at 0.268 ± 0.057 kPa. When we benchmarked this measure of adhesive strength with that of the model DEJ fabricated with cells of individuals with RDEB, the yield stress was significantly lower (0.153 ± 0.064 kPa) consistent with our current mechanistic understanding of RDEB. In summary, a 3D in vitro model DEJ was developed for quantification of mechanical adhesion between epidermal- and dermal-mimicking layers, which can be utilized for assessment of mechanical adhesion of the model DEJ applicable for Epidermolysis Bullosa-associated therapeutics. © 2018 The Authors. Journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3231-3238, 2018.

Rationale: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell–derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. Methods and Results: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell–derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. Conclusions: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix–based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Cardiovascular disease (CVD) is the leading cause of death globally, accounting for 31.5% of deaths worldwide. The widespread (and growing) prevalence of CVD and the inadequacy of the adult human heart to regenerate on its own after a major ischemic event calls for the development of tissue regeneration strategies to facilitate and support cardiomyocyte maturation and function in vivo. This chapter explores regenerative therapies of graded levels of complexity beginning with cell transplantation and ending with a full thickness graft with perfusable vasculature.

The ability to quantify cell migration and invasion is critical in the study of cancer metastasis. Current invasion assays, such as the Boyden Chamber, present difficulties in the measurement of the invasion of cells that are few in number and are intrinsically tied to the cell microenvironment. There exists a need for a three-dimensional invasion assay that is easily reproduced, accessible for most laboratories, and requires no displacement of cells from their original microenvironment. Here we present a simple design for an inverted vertical invasion assay able to assess the invasion capabilities of cells in a three dimensional, extracellular matrix-based environment without displacement from the original culture location. We used the assay to determine the migratory capacity of hybrids between mesenchymal/multipotent stem/stroma cells (MSCs) and breast cancer cells MCF7. These hybrids are formed reliably but rarely (1 in 1,000 cells) and for this reason require an invasion assay that does not involve extensive cell manipulation. Using this assay, we found that MSCs, breast cancer cells, and corresponding fusion products are able to migrate and invade through the extracellular matrix and that hybrids invade in a manner more similar to stromal cells than cancer cells. Thus, this assay can aid the study of the invasive capacity of both cancerous cells and associated fusion hybrids and could augment testing of therapeutic strategies to inhibit metastatic spread.

The cardiac extracellular matrix (cECM) is comprised of proteins and polysaccharides secreted by cardiac cell types, which provide structural and biochemical support to cardiovascular tissue. The roles of cECM proteins and the associated family of cell surface receptor, integrins, have been explored in vivo via the generation of knockout experimental animal models. However, the complexity of tissues makes it difficult to isolate the effects of individual cECM proteins on a particular cell process or disease state. The desire to further dissect the role of cECM has led to the development of a variety of in vitro model systems, which are now being used not only for basic studies but also for testing drug efficacy and toxicity and for generating therapeutic scaffolds. These systems began with 2D coatings of cECM derived from tissue and have developed to include recombinant ECM proteins, ECM fragments, and ECM mimics. Most recently 3D model systems have emerged, made possible by several developing technologies including, and most notably, 3D bioprinting. This chapter will attempt to track the evolution of our understanding of the relationship between cECM and cell behavior from in vivo model to in vitro control systems. We end the chapter with a summary of how basic studies such as these have informed the use of cECM as a direct therapy.

Cardiovascular disease is the global leading cause of death. One route to address this problem is using biomedical imaging to measure the molecules and structures that surround cardiac cells. This cellular microenvironment, known as the cardiac extracellular matrix, changes in composition and organization during most cardiac diseases and in response to many cardiac treatments. Measuring these changes with biomedical imaging can aid in understanding, diagnosing, and treating heart disease. This chapter supports those efforts by reviewing representative methods for imaging the cardiac extracellular matrix. It first describes the major biological targets of ECM imaging, including the primary imaging target of fibrillar collagen. Then it discusses the imaging methods, describing their current capabilities and limitations. It categorizes the imaging methods into two main categories: organ-scale noninvasive methods and cellular-scale invasive methods. Noninvasive methods can be used on patients, but only a few are clinically available, and others require further development to be used in the clinic. Invasive methods are the most established and can measure a variety of properties, but they cannot be used on live patients. Finally, the chapter concludes with a perspective on future directions and applications of biomedical imaging technologies.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Differentiation of adult stem/progenitor cells into functional beta cells to provide a successful autologous cell therapy for Type 1 diabetes patients has not yet been achieved. Progenitor cells in the adult pancreas or liver have been considered potential sources of endocrine beta cells based on their ability to repopulate the organ following injury. Here we describe the isolation and reprogramming of a lineage-committed progenitor population of cells in the human bile duct towards a beta cell fate. These cells, which possess SOX9 as a marker, were able to manifest beta cell characteristics upon ectopic expression of pancreatic transcription factors. The beta cells derived from SOX9+ progenitor cells were also able to ameliorate high blood glucose in diabetic mice. The insulin+ islet-like clusters which developed from this progenitor cell type demonstrate the potential of this approach to generate functional, autologous beta cells.

Although cancer cell fusion has been suggested as a mechanism of cancer metastasis, the underlying mechanisms defining this process are poorly understood. In a recent study, apoptotic cells were newly identified as a type of cue that induces signaling via phosphatidylserine receptors to promote fusion of myoblasts. The microenvironment of breast tumors is often hypoxic, and because apoptosis is greatly increased in hypoxic conditions, we decided to investigate whether the mechanism of breast cancer cell fusion with mesenchymal stem/multipotent stromal cells (MSCs) involves apoptosis. We used a powerful tool for identification and tracking of hybrids based on bimolecular fluorescence complementation (BiFC) and found that breast cancer cells fused spontaneously with MSCs. This fusion was significantly enhanced with hypoxia and signaling associated with apoptotic cells, especially between nonmetastatic breast cancer cells and MSCs. In addition, the hybrids showed a significantly higher migratory capacity than did the parent cells. Taken together, these findings describe a mechanism by which hypoxia-induced apoptosis stimulates fusion between MSCs and breast tumor cells resulting in hybrids with an enhanced migratory capacity that may enable their dissemination to distant sites or metastases. In the long run, this study may provide new strategies for developing novel drugs for preventing cancer metastasis.

The future of regenerative medicine relies on our ability to control stem cell fate in order to produce functional tissues. Stem cells are the preferred cell source for tissue engineering endeavors and regenerative medicine therapies due to their high potency and capacity for expansion. However, their potency also makes them very difficult to control, as they are in a constant state of flux. Therefore, in order to advance research in regenerative medicine, it is necessary to be able to monitor cell state and phenotype both in vitro and in vivo. This review will detail the imaging technologies currently in use to monitor stem cell phenotype, migration, and differentiation. In addition to providing examples of the most recent work in this area, we will also discuss the future of imaging technologies for regenerative medicine, and how current imaging modalities might be utilized to image specific cell functionality in order to track stem cell fate. The research area of imaging stem cells is progressing toward identifying mature and differentiating cells not only by phenotypic markers, but also by visualizing cell function. Many of the cutting-edge modalities detailed in this review have the potential to be harnessed toward this goal.

Evidence suggests that transplanted mesenchymal stem cells (MSCs) can aid recovery of damaged myocardium caused by myocardial infarction. One possible mechanism for MSC-mediated recovery is reprogramming after cell fusion between transplanted MSCs and recipient cardiac cells. We used a Cre/LoxP-based luciferase reporter system coupled to biophotonic imaging to detect fusion of transplanted human pluripotent stem cell-derived MSCs to cells of organs of living mice. Human MSCs, with transient expression of a viral fusogen, were delivered to the murine heart via a collagen patch. At 2 days and 1 week later, living mice were probed for bioluminescence indicative of cell fusion. Cell fusion was detected at the site of delivery (heart) and in distal tissues (i.e., stomach, small intestine, liver). Fusion was confirmed at the cellular scale via fluorescence in situ hybridization for human-specific and mouse-specific centromeres. Human cells in organs distal to the heart were typically located near the vasculature, suggesting MSCs and perhaps MSC fusion products have the ability to migrate via the circulatory system to distal organs and engraft with local cells. The present study reveals previously unknown migratory patterns of delivered human MSCs and associated fusion products in the healthy murine heart. The study also sets the stage for follow-on studies to determine the functional effects of cell fusion in a model of myocardial damage or disease. Significance Mesenchymal stem cells (MSCs) are transplanted to the heart, cartilage, and other tissues to recover lost function or at least limit overactive immune responses. Analysis of tissues after MSC transplantation shows evidence of fusion between MSCs and the cells of the recipient. To date, the biologic implications of cell fusion remain unclear. A newly developed in vivo tracking system was used to identify MSC fusion products in living mice. The migratory patterns of fusion products were determined both in the target organ (i.e., the heart) and in distal organs. This study shows, for the first time, evidence of fusion products at sites distal from the target organ and data to suggest that migration occurs via the vasculature. These results will inform and improve future, MSC-based therapeutics.

Noninvasive biomarkers hold important potential for the characterization and purification of stem cells because the addition of exogenous labels, probes, or reporters, as well as the disruption of cell-cell and cell-extracellular matrix interactions, can unintentionally but dramatically alter stem cell state. We recently showed that intensity of the intrinsically fluorescent metabolite, nicotinamide adenine dinucleotide (NADH), fluctuates predictably with changes in stem cell viability and differentiation state. Here, we use multiphoton flow cytometry developed in our laboratory to rapidly and noninvasively characterize and purify populations of intact stem cell aggregates based on NADH intensity and assessed the differentiation capacity of sorted populations. We found removal of aggregates with NADH intensity indicative of cell death resulted in a remaining population of aggregates significantly more likely to produce beating cardiomyocytes (26% vs. 8%, P

We report Fourier transform infrared spectro-microtomography, a nondestructive three-dimensional imaging approach that reveals the distribution of distinctive chemical compositions throughout an intact biological or materials sample. The method combines mid-infrared absorption contrast with computed tomographic data acquisition and reconstruction to enhance chemical and morphological localization by determining a complete infrared spectrum for every voxel (millions of spectra determined per sample).

Although molecular mechanisms and signaling pathways driving invasion and metastasis have been studied for many years, the origin of the population of metastatic cells within the primary tumor is still not well understood. About a century ago, Aichel proposed that cancer cell fusion was a mechanism of cancer metastasis. This hypothesis gained some support over the years, and recently became the focus of many studies that revealed increasing evidence pointing to the possibility that cancer cell fusion probably gives rise to the metastatic phenotype by generating widespread genetic and epigenetic diversity, leading to the emergence of critical populations needed to evolve resistance to the treatment and development of metastasis. In this review, we will discuss the clinical relevance of cancer cell fusion, describe emerging mechanisms of cancer cell fusion, address why inhibiting cancer cell fusion could represent a critical line of attack to limit drug resistance and to prevent metastasis, and suggest one new modality for doing so.

Methods to generate murine cardiomyocytes from pluripotent stem cells (PSCs) in vitro are resource and time intensive. All current protocols require exogenously provided soluble factors and almost all utilize embryoid body formation to modulate pathways associated with mesoderm specification and cardiomyocyte differentiation. Here, we developed a simple protocol without EBs and without exogenous soluble factors that enabled cardiomyocyte differentiation of a murine induced PSC line based on controlled nutrient deprivation in 2D monolayer cultures. We showed that this protocol reproducibly imposed metabolic stress and consequently modulated active β-catenin levels to yield functional cardiomyocytes. The yield of cardiomyocytes and calcium handling kinetics were comparable to existing approaches. However, this approach did not produce consistent results between murine PSC lines suggesting signaling pathways linking nutrient deprivation to β-catenin activation are not universally conserved and may be a remnant of the parent population from which the induced PSCs were derived.

The promise of cardiac tissue engineering is in the ability to recapitulate in vitro the functional aspects of a healthy heart and disease pathology as well as to design replacement muscle for clinical therapy. Parts of this promise have been realized; others have not. In a meeting of scientists in this field, five central challenges or “big questions” were articulated that, if addressed, could substantially advance the current state of the art in modeling heart disease and realizing heart repair.

Cell fusion can occur between mesenchymal stem cells (MSCs) transplanted to improve cardiac function and cells of the recipient. The therapeutic benefit or detriment of resultant cell hybrids is unknown. Here we augment fusion of transplanted MSCs with recipient cardiac cell types via viral fusogens to determine how cardiac function is impacted. Using a Cre/LoxP-based luciferase reporter system coupled to biophotonic imaging and echocardiography, we found that augmenting fusion with the vesicular stomatitis virus glycoprotein (VSVG) increased the amount of fusion in the recipient mouse heart, but led to diminished cardiac function. Specifically, MSCs transfected with VSVG (MSC-VSVG) had the lowest mean fold increase in fractional area change (FAC) and cardiac output (CO). Although the amount of fusion detected had a strong positive correlation (Pearson) with fractional area change and cardiac output at day 7, this effect was lost by day 28. The decrease in cardiac function seen with MSC-VSVG treatment versus MSC alone or sham treatment was associated with decreased MSC retention, altered immune cell responsiveness and reduced vascularization in the heart. This outcome garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion.

Background information Continued advances in stem cell biology and stem cell transplantation rely on non-invasive biomarkers to characterise cells and stem cell aggregates. The non-invasive quality of such biomarkers is essential because exogenous labels, probes or reporters can unintentionally and dramatically alter stem cell state as can disruption of cell–cell and cell–matrix interactions. Here, we investigate the utility of the autofluorescent metabolite, nicotinamide adenine dinucleotide (NADH), as a non-invasive, intrinsic biomarker of cell death when detected with multi-photon optical-based approaches. To test this possibility, cell death was induced in murine embryoid bodies (EBs) at an early stage (day 3) of differentiation using staurosporine, an ATP-competitive kinase inhibitor of electron transport. Several hours after staurosporine treatment, EBs were stained with a single-colour, live/dead probe. A single-cross-sectional plane of each EB was imaged to detect the fluorescence intensity of the live/dead probe (extrinsic fluorescence) as well as the fluorescence intensity of NADH (intrinsic fluorescence). EBs were assessed at subsequent time points (days 6–12) for the formation of beating areas as an indicator of functional differentiation. Results Statistical comparison indicated a strong positive correlation between extrinsic fluorescence intensity of the live/dead stain and intrinsic fluorescence of NADH, suggesting that the intensity of NADH fluorescence could be used to reliably and non-invasively assess death of cells of EBs. Furthermore, EBs that had high levels of cell death soon after aggregate formation had limited ability to give rise to functional cardiomyocytes at later time points. Conclusions We demonstrate the utility of NADH fluorescence intensity as a non-invasive indicator of cell death in stem cell aggregates when measured using multi-photon excitation. In addition, we show that the degree of stem cell death at early stages of differentiation is predictive for the formation of functional cardiomyocytes.

Electroporation is a common method of gene transfer that has recently been used to efficiently transfect mesenchymal stem (stromal) cells (MSCs); however, the electrical stimulus has the potential to alter cell state. This study examines possible negative effects of electroporation of human embryonic stem cell (hESC)-derived MSCs including, loss of potency or induction of differentiation. Immunofluorescence and PCR were used to quantify protein and RNA expression of CD73 (an MSC marker) and markers of mature mesenchymal cell types following electroporation. The relative fraction of cells expressing CD73 protein was not altered in cells exposed to a 20 ms pulse at 1000 V or 1500 V compared to controls even after three passages, suggesting MSCs retain multipotency following electroporation. In addition, RNA expression of markers indicative of mature cells of bone, fat and cardiac muscle did not differ from unmanipulated controls soon after electroporation. Taken together, these results indicate electroporation under conditions favorable for MSC transfection does not significantly alter stem cell state.

Cancer cell fusion was suggested as a mechanism of metastasis about a century ago. Since then, many additional modes of material transfer (i.e., tunneling nanotubes, and exosomes) to generate cell hybrids have been identified. However, studies documenting spontaneous tumor hybrid formation in vivo as a mechanism that enables metastasis are still lacking. Here, we tested whether spontaneous hybrid formation in vivo contributes to bona fide metastatic tumors. We first used single cell RNASeq to analyze the gene expression profile of spontaneously formed cancer cell-stromal hybrids, and results revealed that hybrids exhibit a clustering pattern that is distinct from either parental cell and suggestive of substantial diversity of individual hybrids. Despite the newly gained diversity, hybrids can retain expression of critical genes of each parental cell. To assess the biological impact of cancer cell hybrids in vivo, we transfected murine mammary tumor cells, isolated from FVB/N-Tg(MMTV-PyVT)634Mul/J mice (PyVT) with Cre recombinase prior to injection to the murine fat pad of FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J mice such that luciferase expression is induced with hybrid formation; luciferase expression was tracked for up to four months. We observed that hybrid formation occurs spontaneously in vivo and that a significantly higher number of hybrids reside in metastases compared to the primary tumor, supporting the possibility that hybrids can emerge from the primary tumor and proliferate to help create a new tumor at a distant site. Additional studies are now warranted to delineate the mechanisms of cancer cell hybrid transit to metastases since drugs to inhibit hybrid formation might prevent metastatic spread.

Detection and tracking of stem cell state are difficult due to insufficient means for rapidly screening cell state in a noninvasive manner. This challenge is compounded when stem cells are cultured in aggregates or three-dimensional (3D) constructs because living cells in this form are difficult to analyze without disrupting cellular contacts. Multiphoton laser scanning microscopy is uniquely suited to analyze 3D structures due to the broad tunability of excitation sources, deep sectioning capacity, and minimal phototoxicity but is throughput limited. A novel multiphoton fluorescence excitation flow cytometry (MPFC) instrument could be used to accurately probe cells in the interior of multicell aggregates or tissue constructs in an enhanced-throughput manner and measure corresponding fluorescent properties. By exciting endogenous fluorophores as intrinsic biomarkers or exciting extrinsic reporter molecules, the properties of cells in aggregates can be understood while the viable cellular aggregates are maintained. Here we introduce a first generation MPFC system and show appropriate speed and accuracy of image capture and measured fluorescence intensity, including intrinsic fluorescence intensity. Thus, this novel instrument enables rapid characterization of stem cells and corresponding aggregates in a noninvasive manner and could dramatically transform how stem cells are studied in the laboratory and utilized in the clinic.

It is generally accepted in immunology that while T and B cells collaborate for the production of antibodies in response to protein antigens, T cells develop and function in the absence of B cells. However, B cell-deficient subjects and mice have unexplained cellular immune defects. Here, we examined the contribution of B cells/Ig to the generation of diversity and function of T cells. Mice lacking B cells and Ig (JH(-/-)) or having oligoclonal B cells (QM) had a profoundly contracted T cell receptor (TCR) Vbeta repertoire: 0.08 and 1.3% of wild type, respectively. Rejection of H-Y-incompatible skin grafts in QM and JH(-/-) mice was significantly delayed (median, 43 and 22 days, respectively) compared to wild-type mice (median, 16 days). Furthermore, reduction of the TCR Vbeta diversity by thymectomy in wild-type mice significantly increased survival of H-Y-incompatible skin grafts, and reconstitution of the T cell diversity in QM mice with Ig Fab fragments significantly decreased survival of the skin grafts. These results indicate that B cells and/or Ig "help" T cells through the generation and maintenance of T cell diversity, improving T cell function. Our results may have important implications for therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post-myeloablative treatments.

For cardiac transplantation in infants, T cells are depleted and the thymus is removed. These manipulations should cause profound defects in the T cell compartment. To test this concept, 20 subjects who underwent cardiac transplantation in infancy and healthy age-matched subjects were studied. The number of T cells in the blood was nearly normal in all subjects 1–10 years after surgery. However, newly generated T cells were undetectable in 10 recipients and 10-fold less than controls in 10, suggesting absence of thymic function. TCRβ chain diversity, measured by a novel technique, was ∼100-fold lower than controls. T cell function, deduced from levels of human herpesvirus 7 and response to hepatitis B immunization, were notably impaired. Yet cardiac transplant recipients were generally free of opportunistic infections. Our findings demonstrate a novel approach to measuring lymphocyte diversity and suggest that understanding how these subjects resist infection could yield important insights into immune fitness.

There is a current need for a small diameter vascular graft due to the limited supply of autogenous grafts and the failure of synthetic grafts due to thrombosis and/or intimal hyperplasia. The use of living cells and tissues to fabricate a small diameter graft (i.e., tissue engineered blood vessel, TEBV) could be useful given the endothelialization potential and biocompatibility benefits of such a graft. However, while sufficient strength has been attained in a TEBV, coordinate compliance has yet to be fine-tuned. In this study we investigate the effects of biological response modifiers, retinoic acid (RA) and ascorbic acid (AA) on TEBV biomechanics as a function of time and subsequently correlate observed RA/AA induced changes in TEBV mechanics with alterations in smooth muscle cell (SMC) biochemistry. TEBVs were constructed using a fibrillar type I collagen network populated by human aortic smooth muscle cells (AoSMC). Following construction this TEBV was treated with 0.3 mM AA and 0.1 mM RA (concentrations found to induce changes in VSMC phenotype). Ultimate tensile stress (UTS), rate of relaxation (RR) and elastic efficiency (EE) of RA/AA treated and untreated TEBVs were measured following 1, 7, 15, 30, 45, and 60 days of treatment. At corresponding time points, the effect of these treatments on collagen and elastin protein synthesis and mRNA expression was examined. RA/AA treated TEBV strength increased and stiffness decreased compared to controls as a function of time. Relative collagen synthesis in treated TEBVs exceeded control levels by nearly two-fold at 15 and 30 days of incubation. RA/AA treated collagen gene expression followed a similar trend. Relative elastin synthesis was also greater in treated TEBVs as compared to untreated TEBVs at 15 and 30 days of incubation and correspondingly elastin mRNA expression was significantly elevated at 15 days of incubation. These data provide evidence that RA/AA treated TEBVs exhibit mechanical properties which more closely mimic those of a native vessel than their untreated counterparts and that changes in extracellular matrix composition and matrix gene expression in the presence of RA/AA treatment may play an important role in the development of said mechanical properties.

Vascular smooth muscle cells (VSMC) influence vessel structure and function during normal development, and in disease states. VSMC interactions with extracellular matrix, via cell surface integrins, play an important role in these processes. A greater understanding of the molecular basis of these interactions is also critical to advances in the field of cardiovascular tissue engineering. This study examined the role of VSMC integrins in the spontaneous compaction and eventual strengthening of a rudimentary tissue-engineered blood vessel (TEBV) consisting of a fibrillar type I collagen network populated by human aortic smooth muscle cells. Using integrin subunit-specific antibodies, we demonstrated that anti-beta1 (Mab13 and P4C10) and anti-alpha2 (P1E6) antibodies that inhibit aortic smooth muscle cell (AoSMC) adhesion to collagen, also significantly inhibit TEBV compaction during the 24-hour period following TEBV construction. However, no difference in the tensile stress of antibody-treated and control TEBVs was observed at this time point. In contrast, 72 hours after construction, the inhibitory effect of anti-integrin antibodies on compaction had been overcome but tensile stress was decreased in TEBVs treated with anti-alpha2/anti-beta1 antibodies when compared to controls. These data provide evidence linking VSMC integrins, specifically the alpha2beta1 integrin, with the initial compaction, as well as, the postcompaction strengthening of the TEBV.

Infants with heart transplants are generally subjected to thymectomy and T cell depletion. In principle, these manipulations should bring about an enduring defect in the structure and function of the T cell compartment. Consistent with that concept, we found that children with heart transplants have profound contraction of the T cell repertoire and higher levels of herpes viruses following infection. Still, these children are generally free of opportunistic infections and other complications expected of those with immunodeficiency. Here we consider these observations and the implications they offer for understanding immune physiology.

Embryoid Bodies (EBs) are large (> 100 μm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 μm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.

The majority of human tumor cells have highly aberrant karyotypes, typically ascribed to errors during tumor cell division, potentially linked to a failure of DNA repair, or telomeric insufficiency. Here we discuss another option, that of cell fusion which can lead to the re-assortment of chromosomes during post-fusion mitosis. The observation of hyperdiploid cells has a long history in cancer genetics, but the concept of cell fusion has been difficult to test in practice. Here, we examine the role of cell fusion during normal development, and relate that to potential cellular fusion partners for primary tumor cells. In particular, we describe the potential for stromal partner fusion during metastatic mobilization. The evidence for genetic and cytoplasmic diversity in heterotypic fusion partners is described, together with the new tools available to help the evaluation of this process as a tumor driver.

The goal of this proposal is to determine whether cell fusion between tumor cells and hematopoietic cells is the precipitating event for breast cancer metastasis and whether viral fusion proteins enable or catalyze this event. If successful, this discovery would dramatically change our approach to breast cancer therapy in the following specific ways. To date we have completed a significant portion of the tasks delineated in Aim 1. First we have optimized protocols for the separation of myeloid and monocyte populations from human mononuclear cell populations. Also we have optimized electroporation conditions for T47D and human mesenchymal stem cell populations. As a result we have been able to conduct our first co-culture experiments to determine whether breast cancer cells fuse spontaneously with hematopoietic cell types. Preliminary results suggest these populations do fuse spontaneously and that fusion products formed in this way can survive several days and are capable of proliferation. In the upcoming months, combinatorial co-cultures with different blood cell subpopulations (myeloid, lymphoid, and monocyte) and healthy or diseased mammary lines will be performed to determine the frequency of fusion between these subpopulations. The effect of hypoxia and hypoglycemia on the frequency of fusion will also be determined. Identified fusion products will then be purified via flow cytometry and assessed for the capacity to migrate and proliferate.

Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cellsin vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphologyin vitro.In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMsin vivoand illustrates how viral fusion proteins might better enable such studies.

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated.

Trafficking of cells between mother and fetus during the course of normal pregnancy is well documented. Similarly, cells are known to travel between twins that share either a placenta (i.e. monozygotic) or associated chorion (i.e. monochorionic). Transferred cells are thought to be channelled via the vessels of the placenta or vascular connections established via the chorion and the long-term presence of these cells (i.e. microchimerism) can have important consequences for immune system function and reparative capacity of the host. Whether cells can be transferred between twins with separate placentas and separate chorions (i.e. no vascular connections between placentas) has not been investigated nor have the biological consequences of such a transfer. In the present study, we tested the possibility of this type of cell transfer by injecting human cord blood-derived cells into a portion of the littermates of swine and probing for human cells in the blood and tissues of unmanipulated littermates. Human cells were detected in the blood of 78% of unmanipulated littermates. Human cells were also detected in various tissues of the unmanipulated littermates, including kidney (56%), spleen (33%), thymus (11%) and heart (22%). Human cells were maintained in the blood until the piglets were sacrificed (8 months after birth), suggesting the establishment of long-term microchimerism. Our findings show that the transfer of cells between fetuses with separate placentas and separate chorions is significant and thus such twins may be subject to the same consequences of microchimerism as monozygotic or monochorionic counterparts.

Cell screening, the process of distinguishing one cell type from another, is particularly essential for stem cells as cell genotype, phenotype and function can change rapidly and unpredictably over time. Initial stem cell characterization efforts were derived primarily from cell biology techniques that were limited in throughput and content and did not typically maintain cellular integrity. More recently, high-throughput techniques including gene or protein arrays, flow cytometry and robotic-based assaying and high-content type techniques including imaging flow cytometry have been developed to more efficiently and comprehensively screen stem cells. However these approaches still rely on sample manipulation to gain information beyond morphology and are incapable of analyzing intact cell aggregates or tissues – structures known to be crucial for the maintenance of stem cell state. To address these limitations, multiphoton laser scanning microscopy, an imaging modality capable of deep sample penetration at high resolution and sensitivity, has been coupled to imaging flow cytometry systems to assay intrinsic fluorescent properties of intact multicell aggregates. Future advances in stem cell screening will likely follow this trend – the coupling of advanced imaging techniques to high throughput/high content modalities – to generate a panel of screening approaches capable of providing not only spatial and temporal information but also physical and chemical properties of cells and their microenvironment in a rapid and noninvasive manner. In this way, stem cell screening might be advanced beyond the identification of cell state to the determination of likely cell function.

Immunotherapy and vaccination for cancer or infection are generally approached by administration of antigen or stimulation of antigen-presenting cells or both. These measures may fail if the treated individual lacks T cells specific for the immunogen(s). We tested another strategy-the generation of new T cells from hematopoietic stem cells that might be used for adoptive immunotherapy. To test this concept, we introduced T cell-depleted human bone marrow cells into fetal swine and tested the swine for human T cells at various times after birth. Human T cells were detected in the thymus and blood of the treated swine. These cells were generated de novo as they contained human T cell receptor excision circles not present in the T cell-depleted bone marrow. The human T cells were highly diverse and included novel specificities capable of responding to antigen presented by human antigen-presenting cells. Our findings constitute a first step in a new promising approach to immunotherapy in which tumor- or virus-specific T cell clones lacking in an individual might be generated in a surrogate host from hematopoietic stem cells of the individual to be treated.

The plasticity and immunomodulatory capacity of mesenchymal stem cells (MSCs) have spurred clinical use in recent years. However, clinical outcomes vary and many ascribe inconsistency to the tissue source of MSCs. Yet unconsidered is the extent of heterogeneity of individual MSCs from a given tissue source with respect to differentiation potential and immune regulatory function. Here we use single-cell RNA-seq to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow. We found genes associated with MSC multipotency were expressed at a high level and with consistency between individual cells. However, genes associated with osteogenic, chondrogenic, adipogenic, neurogenic and vascular smooth muscle differentiation were expressed at widely varying levels between individual cells. Further, certain genes associated with immunomodulation were also inconsistent between individual cells. Differences could not be ascribed to cycles of proliferation, culture bias or other cellular process, which might alter transcript expression in a regular or cyclic pattern. These results support and extend the concept of lineage priming of MSCs and emphasize caution for in vivo or clinical use of MSCs, even when immunomodulation is the goal, since multiple mesodermal (and even perhaps ectodermal) outcomes are a possibility. Purification might enable shifting of the probability of a certain outcome, but is unlikely to remove multilineage potential altogether.

Transplantation of the kidney first became feasible in the early years of the 20th century ([1][1]). Experimental surgeons had recently devised the vascular anastomosis as a way of repairing the cut end of blood vessels, and that advance created the field of vascular surgery ([2,3][2][⇓][3]).

Because organ transplantation is the preferred treatment for organ failure, the demand for human organs for transplantation is large and growing. From this demand, several fields based on new technologies for the replacement or repair of damaged tissues and organs have emerged. These fields include stem cell biology, cloning, tissue engineering and xenotransplantation. Here we evaluate the potential contribution of these to the devising of alternative approaches to organ replacement. We present our vision for the development of two structurally complex organs - the lung and the kidney - based on a 'fusion' of new and established technologies.

The number of patients in need of an organ transplant is increasing, while the number of satisfactory sources of organs has declined in many countries [101]. The resulting shortage of human organs has spurred an urgent effort to investigate alternative therapies, including the use of animal organs, tissues and cells (i.e., xenotransplantation). Advances in genetic engineering have provided essential tools for the development of practical solutions to human disease. The area of xenotransplantation is no exception. In fact, the use of genetic therapies is especially attractive in the transplant setting as it offers an opportunity to manipulate the donor tissue rather than the recipient. This review will describe the obstacles in the clinical application of xenotransplantation and how genetic engineering might be used to address them.

Increasingly, invitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% ± 13.5%, and enrichment ratio of 12.2 ± 8.4 (n = 8), while preserving cell viability, differentiation potential, and long-term function.

Background: Classic reports on lymphocyte development hold that B and T cells develop independently. This concept derives in part from the observation that patients with pure B cell immunodeficiency and hypogammaglobulinemia have a normal thymus and T cell numbers. Our recent findings however challenge this concept. We found that T cell development depends not only on the interaction of T cell precursors with thymic epithelial cells but also on other cells. Here we report that those other cells are B cells. Aims: The purpose of this study was to determine whether B cells drive T cell development and TCR diversification in the thymus. Methods: We compared the number of sub-populations of thymocytes and TCR repertoire diversity in B-cell deficient and B-cell proficient mice and in B cell deficient mice following immunoglobulin (Ig) injections. Total leucocytes numbers were determined with a Coulter counter and numbers of thymocytes sub-populations were calculated by flow cytometry analysis. TCR repertoire diversity was measured by a novel method based on hybridization of TCR Vβ specific cRNA on a gene chip platform. Results: In B-cell deficient mice the number of thymocytes was four times reduced and TCR Vβ chain diversity was up to one million times lower compared with wild type mice. Numbers and diversity were restored by treatment of the mice with gamma globulin (see table). Conclusions: T cell development and diversification is driven by B cells. Mice Number of total thymocytes (mean ± standard deviation) p Value β V TCR diversity of thymocytes (median; min.-max.) p Value C57BL/6 (wild mice) 1.3 x 108 ± 5.1 x 107N=7 4.7 x 106; 1.0 x 105 − 1.1 x 108N=5 JH−/− (B cell immunodeficient mice) 3.1 x 107 ± 1.7 x107N=7 0.002 5.9 x 102; 3.6 x 102 − 1.1 x 103N=5 0.0002 JH−/− treated with Ig 3.9 x 107 ± 1.4 x106N=2 0.20 1.1 x 105; 2.7 x 100 − 7.7 x 105N=4 0.08

The foremost cause of death and disability is organ failure. Failure of the heart, lungs, kidney, and liver is typically addressed by organ transplantation; however, the number of human organs available for this purpose is quite limited. For this reason, some new technologies are being advanced for the treatment of organ failure. This communication summarizes potential limitations of these technologies.

The ability to mount an immune defense against infectious microorganisms and their products, and against tumors is believed to be a direct function of lymphocyte diversity. Because the diversity of lymphocyte receptor genes is >1000-fold more diverse than the entire genome and varies between genetically identical individuals, measuring lymphocyte diversity has been a daunting challenge. We developed a novel technique for measuring lymphocyte diversity directly using gene chips. We reasoned and here demonstrate that the frequency of hybridization of nucleic acids coding for lymphocyte receptors to the oligonucleotides on a gene chip varies in direct proportion to diversity. We applied the technique to detect changes in lymphocyte diversity in mice with known B cell alterations and in persons with known T cell repertoire defects. This approach is the first to provide direct analysis of lymphocyte receptor diversity and should facilitate fundamental study of the adaptive immune system and clinical efforts to assess immunological diseases. In addition, this approach could be more broadly applied, for example to measure diversity of viral quasi-species.

T cell diversity was once thought to depend on the interaction of T cell precursors with thymic epithelial cells. Recent evidence suggests, however, that diversity might arise through the interaction of developing T cells with other cells, the identity of which is not known. In this study we show that T cell diversity is driven by B cells and Ig. The TCR Vβ diversity of thymocytes in mice that lack B cells and Ig is reduced to 6 × 102 from wild-type values of 1.1 × 108; in mice with oligoclonal B cells, the TCR Vβ diversity of thymocytes is 0.01% that in wild-type mice. Adoptive transfer of diverse B cells or administration of polyclonal Ig increases thymocyte diversity in mice that lack B cells 8- and 7-fold, respectively, whereas adoptive transfer of monoclonal B cells or monoclonal Ig does not. These findings reveal a heretofore unrecognized and vital function of B cells and Ig for generation of T cell diversity and suggest a potential approach to immune reconstitution.