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The heart is a highly complex, multicellular solid organ with energy-demanding processes that require a dense vascular network, extensive cell-cell interactions, and extracellular matrix (ECM)-mediated crosstalk among heterogeneous cell populations. Here, we describe the regeneration of left ventricular (LV) wall using decellularized whole rabbit heart scaffolds recellularized exclusively with human induced pluripotent stem cell-derived endothelial cells, cardiomyocytes, and other cardiac cell types. Cells were sequentially delivered to the scaffold using an optimized endothelial cell:cardiomyocyte media. Macroscopic assessment after 60 days showed that the LV wall of recellularized hearts was anatomically restored to full thickness from base to apex and endocardium to epicardium. Histologic analysis of the recellularized LV wall revealed a heterogeneous pool of cardiac cells containing aligned cardiac troponin T-positive cells in close contact with ECM; vessels varied from large artery-like, surrounded by smooth muscle actin+ cells, to capillary-like. Vessel patency was demonstrated after perfusion of recellularized hearts transplanted into the femoral artery bed of a pig. The construct exhibited visible beating and responded to chronotropic drug administration. These results demonstrate the ability to tissue engineer a vascularized, full-thickness LV wall with an unparalleled level of microanatomical organization and multicellular composition, using decellularized ECM and human cardiomyocytes, endothelial cells, and other cardiac cell types. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (ECM) is a bioactive template for tissue engineering, but recellularizing acellular whole heart scaffolds is challenging. Here, we successfully revascularized and repopulated a large, full-thickness portion of a ventricle using human induced pluripotent stem cell-derived endothelial and cardiac cells. At 60 days, histologic studies showed that the microanatomical organization and cellular composition of this region was similar to that of the native heart. The recellularized heart showed visible beating and responded appropriately to heartbeat-altering drugs. Vessels surrounded by smooth muscle cells and endothelial cells supported blood flow through the vessels of a recellularized heart that was surgically connected to a pig femoral artery. These findings move this approach closer to the possibility of clinical translation.

During fetal development, cardiomyocytes switch from glycolysis to oxidative metabolism to sustain the energy requirements of functional cells. State-of-the-art cardiac differentiation protocols yield phenotypically immature cardiomyocytes, and common methods to improve metabolic maturation require multistep protocols to induce maturation only after cardiac specification is completed. Here, we describe a maturation method using ventricle-derived decellularized extracellular matrix (dECM) that promoted early-stage metabolic maturation of cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). Chemically and architecturally preserved particles (45–500 μm) of pig ventricular dECM were added to hiPSCs at the start of differentiation. At the end of our maturation protocol (day 15 of cardiac differentiation), we observed an intimate interaction between cardiomyocytes and dECM particles without impairment of cardiac differentiation efficiency (approx. 70% of cTNT+). Compared with control cells (those cultured without pig dECM), 15-day-old dECM-treated cardiomyocytes demonstrated increased expression of markers related to cardiac metabolic maturation, MAPK1, FOXO1, and FOXO3, and a switch from ITGA6 (the immature integrin isoform) to ITGA3 and ITGA7 (those present in adult cardiomyocytes). Electrical parameters and responsiveness to dobutamine also improved in pig ventricular dECM-treated cells. Extending the culture time to 30 days, we observed a switch from glucose to fatty acid metabolism, indicated by decreased glucose uptake and increased fatty acid consumption in cells cultured with dECM. Together, these data suggest that dECM contains endogenous cues that enable metabolic maturation of hiPSC-CMs at early stages of cardiac differentiation.

Processes that activate the immune system during lung transplantation can lead to primary graft dysfunction (PGD) or allograft rejection.We analyzed cytokine expression profiles after reperfusion and allograft outcomes in a cohort of patients (n=59) who underwent lung transplantation off-pump (n=26), with cardiopulmonary bypass (CPB; n=18), or with extracorporeal membrane oxygenation (ECMO; n=15). Peripheral blood was collected from patients at baseline and at 6 and 72 hours after reperfusion. To adjust for clinical differences between groups, we utilized a linear mixed model with overlap weighting.PGD3 was present at 48 or 72 hours after reperfusion in 7.7% (2/26) of off-pump cases, 20.0% (3/15) of ECMO cases, and 38.9% (7/18) of CPB cases (p=0.04). The ECMO and CPB groups had greater reperfusion-induced increases in MIP-1B, IL-6, IL-8, IL-9, IL1-ra, TNF-alpha, RANTES, eotaxin, IP-10, and MCP-1 levels than did the off-pump group. Cytokine expression profiles after reperfusion were not significantly different between ECMO and CPB groups.Our data suggest that, compared with an off-pump approach, the intraoperative use of ECMO or CPB during lung transplantation is associated with greater reperfusion-induced cytokine release and graft injury.

New robust and reproducible differentiation approaches are needed to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes of specific subtypes in predictable quantities for tissue-specific disease modeling, tissue engineering, and eventual clinical translation. Here, we assessed whether powdered decellularized extracellular matrix (dECM) particles contained chamber-specific cues that could direct the cardiac differentiation of human iPSCs toward an atrial phenotype. Human hearts were dissected and the left ventricle (LV) and left atria (LA) were isolated, minced, and decellularized using an adapted submersion decellularization technique to generate chamber-specific powdered dECM. Comparative proteomic analyses showed chamber-specific dECM segregation, with atrial- and ventricle-specific proteins uniquely present in powdered dECM-hA and dECM-hV, respectively. Cell populations differentiated in the presence of dECM-hA showed upregulated atrial molecular markers and a two-fold increase in the number of atrial-like cells as compared with cells differentiated with dECM-hV or no dECM (control). Finally, electrophysiological data showed an increase in action potentials characteristic of atrial-like cells in the dECM-hA group. These findings support the hypothesis that dECM powder derived from human atria retained endogenous cues to drive cardiac differentiation toward an atrial fate.

INTRODUCTION: Recellularization of organ decellularized extracellular matrix (dECM) offers a potential solution for organ shortage in allograft transplantation. Cell retention rates have ranged from 10 to 54% in varying approaches for reseeding cells in whole organ dECM scaffolds. We aimed to improve recellularization by using soluble gelatin as a cell carrier to deliver endothelial cells to the coronary vasculature and cardiomyocytes to the parenchyma in a whole decellularized rat heart. METHODS: Rat aortic endothelial cells (RAECs) were perfused over decellularized porcine aorta in low (1%) and high (5%) concentrations of gelatin to assess attachment to a vascular dECM model. After establishing cell viability and proliferation in 1% gelatin, we used 1% gelatin as a carrier to deliver RAECs and neonatal rat cardiomyocytes (NRCMs) to decellularized adult rat hearts. Immediate cell retention in the matrix was quantified, and recellularized hearts were evaluated for visible contractions up to 35 days after recellularization. RESULTS: We demonstrated that gelatin increased RAEC attachment to decellularized porcine aorta; blocking integrin receptors reversed this effect. In the whole rat heart gelatin (1%) increased retention of both RAECs and NRCMs respectively, compared with the control group (no gelatin). Gelatin was associated with visible contractions of NRCMs within hearts (87% with gelatin vs. 13% control). CONCLUSIONS: Gelatin was an effective cell carrier for increasing cell retention and contraction in dECM. The gelatin-cell-ECM interactions likely mediated by integrin. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-020-00634-z) contains supplementary material, which is available to authorized users.

To expand the application of perfusion decellularization beyond isolated single organs, we used the native vasculature of adult and neonatal rats to systemically decellularize the organs of a whole animal in situ. Acellular scaffolds were generated from kidney, liver, lower limb, heart‐lung system, and a whole animal body, demonstrating that perfusion decellularization technology is applicable to any perfusable tissue, independent of age. Biochemical and histological analyses demonstrated that organs and organ systems (heart‐lung pair and lower limb) were successfully decellularized, retaining their extracellular matrix (ECM) structure and organ‐specific composition, as evidenced by differences in organ‐specific scaffold stiffness. Altogether, we demonstrated that organs, organ systems and whole animal bodies can be perfusion decellularized while retaining ECM components and biomechanics., Representative images from systemic perfusion decellularization of an adult female whole rat body. Histological staining of excised organs and organs systems shows the absence of cells yet maintenance of tissue‐specific characteristics.

Background Primary graft dysfunction (PGD) is the most important determinant of morbidity and mortality after lung transplantation, but its definition has evolved over the past decade. The implications of this refinement in clinical definition have not been evaluated. In this single-center study, we compared PGD incidence, risk factors, and outcomes using the 2005 and the updated-2016 International Society of Heart and Lung Transplantation guidelines for PGD grading in lung transplant patients. Methods In this retrospective study, we extracted data from the medical records of 127 patients who underwent lung transplantation between 1/1/2016-12/31/2018. PGD was defined as PGD3 present at 48 and/or 72 hours post-reperfusion. We used the 2005 and the updated 2016 guidelines to assess clinical risk factors, outcomes, and baseline biomarkers for PGD. Results On the basis of the 2016 and 2005 guidelines, we identified PGD in 37% and 26% of patients, respectively. PGD was significantly associated with extracorporeal life support, large body mass index, and restrictive lung disease using the 2016 but not the 2005 guidelines. Based on the 2016 guidelines, pretransplant levels of several biomarkers were associated with PGD; using the 2005 guidelines, only increased interleukin-2 levels were significantly associated with PGD. No preoperative biomarkers were associated with PGD using either guidelines after adjusting for clinical variables. Postoperative morbidity and 1-year mortality were similar regardless of guidelines used. Conclusions Our findings suggest that refinements in the PGD scoring system have improved the detection of graft injury and associated risk factors without changing its ability to predict postoperative morbidity and mortality.

Establishment of expansion systems that are synthetic, defined, and stable will help accelerate the translation of human pluripotent stem cells (hPSCs). This chapter describes strategies to eliminate feeder cell layer systems for PSC expansion based on studies in cell–extracellular matrix (ECM) interactions and recent developments in PSC technologies. We survey the literature describing laminin (both laminin-511 and laminin-521) as a feeder-free culture substrate for hPSC expansion. We describe our group’s findings that the use of laminin for bioreactor-based large-scale expansion of induced pluripotent stem cells (iPSCs) is user-friendly, reproducible, and can be scaled-up. Toward the development of a tunable, xeno-free, coating solution that preserves pluripotency, we posit that laminin-based strategies for large-scale expansion of hPSCs will be critical for the optimization of basic research applications and ultimately for the realization of the therapeutic promise of these cells.

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.

No effective medical treatment exists for heart failure with preserved ejection fraction (HFpEF), accounting for approximately half of all heart failure cases. The elevated passive myocardial stiffness in HFpEF is attributed to a combination of alterations in the extracellular matrix (ECM) collagen content and modifications in the sarcomeric protein titin. Here, we propose polylaminin, a biomimetic polymer of laminin, as a promising approach for manipulating the titin isoform shift and phosphorylation in cardiomyocytes. Exploring the pleiotropic effects of polylaminin may be a novel strategy for alleviating symptoms in HFpEF’s multifactorial pathophysiology.

Mesenchymal stromal cells derived from bone marrow (BM-MSCs) have been considered promisingcandidates for regenerative medicine therapies for more than 20 years, but to date cardiovascular clinicaltrials of MSC-based therapies have demonstrated only modest improvement. However, the mechanismsunderlying why some patients improve and others do not remain undefined. Although MSC survival aftertransplantation remains a challenge, several studies have reported that BM-MSC-mediated paracrineeffects and delivery of exosomes may be therapeutically relevant. We hypothesized that MSC-derivedexosomes from patients with cardiac functional improvement contain factors that promote cell survival,proliferation, and cardiac protection. Here, we compared exosomes (derived from MSCs that had beenexpanded from BM-MNCs used for autologous transplantation) from two cohorts of disease- and age-matched consented patients with ischemic cardiomyopathy enrolled in the FOCUS-CCTRN trial—thosewith improved (cohort1, n=3) or worsened (cohort2, n=3) functional outcome in three primary endpoints:left ventricular (LV) ejection fraction, LV end-systolic volume, and maximal oxygen consumption.MicroRNA-seq analysis identified 16 upregulated and 12 downregulated miRNAs in cohort 1 whencompared to cohort 2. Using DIANA-Mir Path v.3.0, we identified 3,522 experimentally validated genetargets. Gene set enrichment analysis identified enrichment for fatty acid biosynthesis and metabolism,Hippo signaling pathway, protein processing in endoplasmic reticulum, adherent junction, cell cycle,endocytosis, RNA transport and degradation, estrogen-signaling pathway, regulation of actincytoskeleton, Prolactin signaling pathway, Focal adhesion, Notch-signaling pathway, p53-signalingpathway, insulin-signaling pathway and HIF-1-signaling pathway in the targets of upregulated miRNAs.The downregulated miRNAs were related to extracellular matrix-receptor interaction, protein processingin endoplasmic reticulum, cytokine-cytokine receptor interaction and antigen processing andpresentation. Thus, the identified miRNAs differentially expressed in patients with an improved functionaloutcome that may play a role in the molecular mechanisms that underlie in cardiac repair in patients withischemic cardiac disease. Moreover, these finding indicate that not all autologous cell products may becompetent therapy candidates for cardiac repair These results warrant further investigation to confirmthe effects of the identified differences on the target genes, which could improve the prognosis and unveilnew therapeutic approaches.

Extracellular matrix (ECM) can directly modulate cell proliferation, migration and differentiation by mediating diverse growth factors and signaling interactions. Protocols for cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) that recapitulate cardiac development frequently result in a mixed cardiac cell population dominated overwhelmingly by ventricular-like cells. Utilizing the inherent biological capabilities of decellularized ECM (dECM) from human myocardium, we developed a method for committing human iPSCs to an atrial-like cell phenotype. We employed a modified decellularization method to generate small particles (125-500 μm) of human atrial and ventricular dECM. The particles presented a fractal dimension (1.63 and 1.71) that suggested self-similarity across particle sizes of both atrial and ventricular dECM. Quantifications of DNA (3.37±0.50 and 2.77±0.62% of cadaveric), GAG (0.44±0.08 and 0.59±0.13 μg/mg), and SDS (2.46±1.20 and 2.91±2.53 μg/mg) validated the absence of difference of atrial and ventricular dECM. Proteomic profiling revealed dECM chamber-specific clustered populations. Ventricular and atrial dECM segregated into ventricular and atrial parts based on component 1 (19.5%) and component 2 (13.9%). A total of 14% of atrial proteins were matrisome atrial-related and 13% of ventricle proteins were matrisome ventricular-related. Myocytes differentiated in the presence of atrial dECM showed similar differentiation efficiency (66.6±10.2 vs 65.5±12.7% of cTNT) and, importantly, increased atrial markers, as confirmed by qPCR (SLP and COUPF-I) and flow cytometry (43.5%±12.7% vs 23.9%±10.8% of MLC2a) in comparison to control. We observed an increase in atrial cells (38.4% vs 14.8%) by action potential duration (APD), with statistical differences in cAPD10 (57.1±20.2 vs 104.4±48.7 ms) and cAPD20 (76.2±22 vs 126±47.4 ms). Altogether, we demonstrate that human atrial ECM retains cues to drive cardiac differentiation to an atrial fate, doubling the number of atrial cells with a functional atrial phenotype. These findings are a critical step toward generating sufficient quantities of atrial cells, which can be used for chamber-specific cardiac disease modeling and drug development.

Bioengineering a solid organ for organ replacement is a growing endeavor in regenerative medicine. Our approach – recellularization of a decellularized cadaveric organ scaffold with human cells – is currently the most promising approach to building a complex solid vascularized organ to be utilized in vivo, which remains the major unmet need and a key challenge. The 2008 publication of perfusion-based decellularization and partial recellularization of a rat heart revolutionized the tissue engineering field by showing that it was feasible to rebuild an organ using a decellularized extracellular matrix scaffold. Toward the goal of clinical translation of bioengineered tissues and organs, there is increasing recognition of the underlying need to better integrate basic science domains and industry. From the perspective of a research group focusing on whole heart engineering, we discuss the current approaches and advances in whole organ engineering research as they relate to this multidisciplinary field’s 3 major pillars: organ scaffolds, large numbers of cells, and biomimetic bioreactor systems. The success of whole organ engineering will require optimization of protocols to produce biologically-active scaffolds for multiple organ systems, and further technological innovation both to produce the massive quantities of high-quality cells needed for recellularization and to engineer a bioreactor with physiologic stimuli to recapitulate organ function. Also discussed are the challenges to building an implantable vascularized solid organ.

Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.

Cardiovascular disease remains the leading cause of death worldwide, provoking the need for alternative approaches to repairing the diseased or injured heart. The cardiovascular tissue engineering field emerged to develop therapeutic options for structural, functional, and vascular heart diseases, with an overarching goal of building functional cardiac and vascular tissue constructs that can restore or recapitulate healthy heart function. Since its inception, the intensive multidisciplinary nature of cardiovascular tissue engineering has revolutionized our understanding of cardiovascular biology and marshaled advances in both cell and scaffold technologies. The field initially focused on repair-oriented interventions, such as cell-based therapy, vascular grafts, bioprosthetic heart valves, and cardiac patches, but more recently viable cardiac wall and even organ replacements have been proposed. Although definitive repair of the heart remains elusive, today the use of tissue-engineered products has become an option in many clinical scenarios, and even the generation of bioartificial heart replacements has moved from fantasy to potential reality. Here, we discuss current cardiovascular tissue engineering approaches and therapies, the pathway(s) to approval, and how the field may progress in the near future. For the purpose of this chapter, tissue engineering is defined as the use of a combination of cells, scaffolds (both synthetic and biologic), and engineering principles for the formation of new viable tissues/organs for cardiac or vascular medical use.

Cardiovascular disease is an important public health problem. Heart transplantation remains the definitive treatment for patients with end-stage heart failure (ESHF), but the availability of donor hearts falls short of the need. Although the use of mechanical devices has emerged as an alternative solution, these devices have several limitations. Tissue engineering techniques that utilize cells and regenerative medicine for the treatment of various heart diseases including ESHF have shown promise as new approaches in cardiovascular research. For the reconstruction of new tissues, scaffolds (i.e., biomaterials to be used as supports), cells, and appropriate growth factors are needed. Because the biomaterial plays an important role in the attachment of human cells and their functional integrity, generating an ideal scaffold is the greatest challenge in tissue engineering. A decellularized heart composed of native extracellular matrix has been shown to offer a complex, unique, and natural scaffold that provides both physical and chemical cues required for cardiac function. In this chapter, the key steps of reconstructing a whole heart from a decellularized scaffold are described, including procedures before decellularization (e.g., perfusion methods), during decellularization (e.g., monitoring of flow dynamics and outflow turbidity and viscosity), and after decellularization (e.g., disinfectant treatments, biomechanical and microstructural property evaluation). Remaining challenges in the field of cardiac regenerative research are also discussed.

Induced pluripotent stem cell (iPSC) line were generated from erythroblasts of a Brazilian patient with familiar form of amyotrophic lateral sclerosis (ALS). NGS analysis demonstrated that patient carried a mutation in SOD1 gene, as well as a deletion in FUS gene. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC) was used to generate the cell lines. The iPSCs express pluripotency markers, have normal karyotype and differentiated spontaneously in the three germ layers. The expression of Sendai virus was lost in all iPSC lines after 15 passages.

Tissue engineering demands the development of scaffolds that mimic natural extracellular matrices (ECM). Despite the success in obtaining synthetic interstitial ECM, the production of an artificial basement membrane (BM), the specialized thin sheet of ECM that is pivotal for the functional organization of most tissues and internal organs, is still not achieved. With the long-term aim of developing a flat BM-like structure here we investigated the behavior of acid-soluble Col IV during simultaneous assembly with laminin (LM) in acidic conditions. The underlying rationale was the previously observed phenomenon of acid-triggered LM polymerization, giving rise to biomimetic polylaminin (polyLM) that can be adsorbed on the substrate. Unexpectedly, we found that Col IV (that does not polymerize in acidic conditions) readily incorporated into the polyLM layer, forming a network that mimics to a great extent the characteristic polygonal morphology of single polyLM observable at micrometric scale. Scanning calorimetry and light scattering measurements supported the notion that polyLM and Col IV could directly interact. The biological properties of the proposed artificial BM-like structure were characterized using human keratinocytes (HACAT) and umbilical vein endothelial cells (HUVEC). HACAT formed stratified cell layers on the hybrid polyLM/Col IV layer, but not on Matrigel, nor on LM or Col IV alone, while HUVEC improved cortical F-actin and tight juctions organization on polyLM/Col IV. Thus, the proposed artificial BM reproduces not only morphological but also some functional properties of the natural BM. STATEMENT OF SIGNIFICANCE: Basement membranes (BMs) are flat biological matrices separating tissue compartments in the body. Their peculiar sheet-like structure is thought to result from the association of two independent protein networks of laminin and collagen IV. While pursuing the development of an artificial BM, we found that, when mixed with acid-induced polymerized laminin, collagen IV immediately conformed to the laminin shape. This implies that the protein networks may not be independently assembled as believed so far, but instead that laminin may command the assembly of collagen IV. Our hybrid matrix was structurally more stable than the commercial BM extract Matrigel and, unlike the latter, supported in vitro formation of a stratified layer of keratinocytes that approximated the organization of the natural epidermis.

Laminin-111 (Ln-1), an extracellular matrix (ECM) glycoprotein found in the basement membrane of mammary gland epithelia, is essential for lactation. In mammary epithelial cells (MECs), dystroglycan (Dg) is believed to be necessary for polymerization of laminin-111 into networks., thus we asked whether correct polymerization could compensate for Dg loss. Artificially polymerized laminin-111 and the laminin-glycoprotein mix Matrigel, both formed branching, spread networks with fractal dimensions from 1.7 to 1.8, whereas laminin-111 in neutral buffers formed small aggregates without fractal properties (a fractal dimension of 2). In Dg knockout cells, either polymerized laminin-111 or Matrigel readily attached to the cell surface, whereas aggregated laminin-111 did not. In contrast, polymerized and aggregated laminin-111 bound similarly to Dg knock-ins. Both polymerized laminin-111 and Matrigel promoted cell rounding, clustering, formation of tight junctions, and expression of milk proteins, whereas aggregated Ln-1 did not attach to cells or promote functional differentiation. These findings support that the microstructure of Ln-1 networks in the basement membrane regulates mammary epithelial cell function.

There are few existing methods for shortening the decellularization period for a human-sized whole-liver scaffold. Here, we describe a protocol that enables effective decellularization of the liver obtained from pigs weigh 120 ± 4.2 kg within 72 h. Porcine livers (approx. 1.5 kg) were decellularized for 3 days using a combination of chemical and enzymatic decellularization agents. After trypsin, sodium deoxycholate, and Triton X-100 perfusion, the porcine livers were completely translucent. Our protocol was efficient to promote cell removal, the preservation of extracellular matrix (ECM) components, and vascular tree integrity. In conclusion, our protocol is efficient to promote human-sized whole-liver scaffold decellularization and thus useful to generate bioengineered livers to overcome the shortage of organs.

Laminin-111, an extracellular matrix (ECM) glycoprotein found in the basement membrane of mammary gland epithelia, is essential for lactation. In mammary epithelial cells, dystroglycan (Dg) is believed to be necessary for polymerization of laminin-111 into networks, thus we asked whether correct polymerization could compensate for Dg loss. Artificially polymerized laminin-111 and the laminin-glycoprotein mix Matrigel, both formed branching, spread networks with fractal dimensions from 1.7-1.8, whereas laminin-111 in media formed small aggregates without fractal properties (a fractal dimension of 2). In Dg knockout cells, either polymerized laminin-111 or Matrigel readily attached to the cell surface, whereas aggregated laminin-111 did not. In contrast, polymerized and aggregated laminin-111 bound similarly to Dg knock-ins. Both polymerized laminin-111 and Matrigel promoted cell rounding, clustering, formation of tight junctions, and expression of milk proteins, whereas aggregated Ln-1 did not attach to cells or promote functional differentiation.HighlightsLaminin assembles into a fractal network when in presence of either the cell surface receptor dystroglycan or acidic glycoproteins or an acidic buffer.When this microstructure is recreated with an acidic treatment, laminin binds readily to dystroglycan null cells and induces functional differentiation of mammary epithelial cells.

Cardiovascular diseases are the primary cause of death worldwide. The natural evolution of heart disease leads to end-stage heart failure, when organ replacement becomes the only therapeutic option. However, heart transplantation is limited by the availability of donor organs, as well as the possibility of acute rejection and complications resulting from immunosuppressive therapy. As an alternative to transplants, the tissue engineering of whole organs—in particular, of autologous organs—has emerged as a promising therapeutic option. The process involves the construction and maintenance of tissues and organs in vitro by using natural or synthetic biomaterials as a scaffold. A decellularized whole heart, which is composed of extracellular matrix (ECM), proteins, and sugars, is a natural choice for a biologic cardiac scaffold. If generated properly, a decellularized scaffold retains the structure and components of the ECM with minimal damage, thus maintaining the intrinsic mechanical and geometric properties of the organ that are essential for successful recellularization. Engineered whole organs have the potential to alleviate the shortage of donor organs and to reduce the occurrence of rejection associated with transplantation because they can be generated with a recipient's own cells. The use of a natural ECM has emerged in part because synthetic matrices can be immunogenic and can undergo toxic degradation and trigger an inflammatory reaction. In this chapter, we will identify the critical components of the native human heart and present current knowledge regarding the generation of tissue-engineered whole hearts, with a focus on the use of decellularized ECM scaffolds.

In vitro-expanded bone marrow stromal cells (BMSCs) have long been proposed for the treatment of complex bone-related injuries because of their inherent potential to differentiate into multiple skeletal cell types, modulate inflammatory responses, and support angiogenesis. Although a wide variety of methods have been used to expand BMSCs on a large scale by using good manufacturing practice (GMP), little attention has been paid to whether the expansion procedures indeed allow the maintenance of critical cell characteristics and potency, which are crucial for therapeutic effectiveness. Here, we described standard procedures adopted in our facility for the manufacture of clinical-grade BMSC products with a preserved capacity to generate bone in vivo in compliance with the Brazilian regulatory guidelines for cells intended for use in humans. Bone marrow samples were obtained from trabecular bone. After cell isolation in standard monolayer flasks, BMSC expansion was subsequently performed in two cycles, in 2- and 10-layer cell factories, respectively. The average cell yield per cell factory at passage 1 was of 21.93±12.81×106 cells, while at passage 2, it was of 83.05±114.72×106 cells. All final cellular products were free from contamination with aerobic/anaerobic pathogens, mycoplasma, and bacterial endotoxins. The expanded BMSCs expressed CD73, CD90, CD105, and CD146 and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Most importantly, nine out of 10 of the cell products formed bone when transplanted in vivo. These validated procedures will serve as the basis for in-house BMSC manufacturing for use in clinical applications in our center.

The number of high-quality cells required for engineering an adult human-sized bioartificial organ is greater than one billion. Until the emergence of induced pluripotent stem cells (iPSCs), autologous cell sources of this magnitude and with the required complexity were not available. Growing this number of cells in a traditional 2D cell culture system requires extensive time, resources, and effort and does not always meet clinical requirements. The use of a closed cell culture system is an efficient and clinically applicable method that can be used to expand cells under controlled conditions. We aimed to use the Quantum Cell Expansion System (QES) as an iPSC monolayer-based expansion system. Human iPSCs were expanded (up to 14-fold) using the QES on two different coatings (laminin 521 (LN521) and vitronectin (VN)), and a karyotype analysis was performed. The cells were characterized for spontaneous differentiation and pluripotency by RT-PCR and flow cytometry. Our results demonstrated that the QES provides the necessary environment for exponential iPSC growth, reaching 689.75 × 106 ± 86.88 × 106 in less than 7 days using the LN521 coating with a population doubling level of 3.80 ± 0.19. The same result was not observed when VN was used as a coating. The cells maintained normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DPPA4, NODAL, TDGFb, TERT3, and GDF), and expressed high levels of OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T), and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR with both coating systems. We conclude that the QES maintains the stemness of iPSCs and is a promising platform to provide the number of cells necessary to recellularize small human-sized organ scaffolds for clinical purposes.

Engineering a bioartificial heart has become a possibility in part because of the regenerative medicine approaches to repairing or replacing damaged organs that have evolved over the past two decades. With the advent of inducible pluripotent stem cell technology, it is now possible to generate personalized cells that make the concept of autologous tissue engineering imaginable. Scaffolds that provide form, function, and biological cues to cells likewise potentially enable the engineering of biocompatible vascularized solid organs. Decellularized organs or tissue matrices retain organ complexity and structure at the macro and micro scales, contain biologically active molecules that support cell phenotype and function, and are vascularized allowing full thickness tissue generation. There is also dynamic reciprocity between the extracellular matrix and cells, which does not occur with synthetic scaffolds and allows both to evolve as functional need changes, making it a unique scaffold. Yet, building a whole heart from decellularized scaffolds and cells requires delivering hundreds of billions of multiple types of cardiac cells appropriately and providing a milieu where they can survive and mature. We propose a novel type of in vivo organ engineering utilizing pre-clinical models where decellularized hearts are heterotopically transplanted with the intent to harness the capability of the body to at least in part repopulate the scaffold. By adding load and electrical input, possibly via temporary mechanical assistance, we posit that vascular and parenchymal cell maturation can occur. In this study, we implanted porcine decellularized hearts acutely and chronically in living recipients in a heterotopic position. We demonstrated that the surgical procedure is critical to prevent coagulation and to increase graft patency. We also demonstrated that short-term implantation promotes endothelial cell adhesion to the vessel lumens and that long-term implantation also promotes tissue formation with evidence of cardiomyocytes and endothelial cells present within the graft. Utilizing endogenous repair capabilities of the recipient in response to a naked ECM, we allowed the transplanted scaffold to direct host cells-both organizationally and functionally. Thus, the scaffold provided necessary cues for cell organization and remodeling within the transplanted organ. Future work would involve culturing partially recellularized engineered organs in bioreactors where mechanical and electrical stimulation can be controlled to promote organ development and then transplanting these after a minimal level of maturation has been achieved.

Polylaminin (polyLM) is a flat biomimetic polymer of laminin capable of promoting axonal growth both in vitro and in vivo. It is assembled in a cell-free system when laminin 111 is incubated in acidic pH, whereas incubation in neutral buffer leads to the formation of bulky and irregular polymers (LM). In the present work, we compared the behaviors of cells isolated from the P1 rat retina on polyLM and LM. PolyLM induced cellular spreading and the outgrowth of neurites in contact with the substrate, whereas LM led to the formation of large clusters of cells, with neurites growing only inward. After 24 hr in culture, the number of cells on polyLM increased threefold, and this increase was inhibited by 60% in the presence of the PKA inhibitor H89 and by 41% in the presence of the PKC inhibitor chelerythrine chloride, whereas both inhibitors abolished neuritogenesis. Neither the cell number nor the outgrowth of neurites was affected by the ERK1/2 inhibitor PD98059 on polyLM. On the other hand, PD98059 was able to reduce the cell number on LM, whereas the other inhibitors were not. Immunostaining of P1 retina with an antilaminin antibody revealed that the protein was expressed not only at its inner surface but also within the neuroblast layer in close contact with individual cells. Our results indicate that, when provided in its active polymerized form, laminin can influence both neuritogenesis and proliferation of retinal cells.