Priming with oncolytic adenovirus followed by anti-PD-1 and paclitaxel treatment leads to improved anti-cancer efficacy in the 3D TNBC model.

Triple-negative breast cancer (TNBC) is considered one of the most incurable malignancies due to its clinical characteristics, including high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Therefore, it remains a critical unmet medical need. On the other hand, poor delivery efficiency continues to reduce the efficacy of anti-cancer therapeutics developed against solid tumours using various strategies, such as genetically engineered oncolytic vectors used as nanocarriers. The study was designed to evaluate the anti-tumour efficacy of a novel combinatorial therapy based on oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with an anti-PD-1 (pembrolizumab) and paclitaxel (PTX). Here, we first tested the antineoplastic effect in two-dimensional (2D) and three-dimensional (3D) breast cancer models in MDA-MB-231, MDA-MB-468 and MCF-7 cells. Then, to further evaluate the efficacy of combinatorial therapy, including immunological aspects, we established a three-dimensional (3D) co-culture model based on MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) to create an integrated system that more closely mimics the complexity of the tumour microenvironment and interacts with the immune system. Treatment with OV as a priming agent, followed by pembrolizumab and then paclitaxel, was the most effective in reducing the tumour volume in TNBC co-cultured spheroids. Further, T-cell phenotyping analyses revealed significantly increased infiltration of CD8+, CD4+ T and Tregs cells. Moreover, the observed anti-tumour effects positively correlated with the level of CD4+ T cell infiltrates, suggesting the development of anti-cancer immunity. Our study demonstrated that combining different immunotherapeutic agents (virus, pembrolizumab) with PTX reduced the tumour volume of the TNBC co-cultured spheroids compared to relevant controls. Importantly, sequential administration of the investigational agents (priming with the vector) further enhanced the anti-cancer efficacy in 3D culture over other groups tested. Taken together, these results support further evaluation of the virus in combination with anti-PD-1 and PTX for the treatment of triple-negative breast cancer patients. Importantly, further studies with in vivo models should be conducted to better understand the translational aspects of tested therapy.

Culture of Hoffa fat pad mesenchymal stem/stromal cells on microcarrier suspension in vertical wheel bioreactor for extracellular vesicle production.

BACKGROUND: Mesenchymal stromal cells (MSCs) are increasingly employed in regenerative medicine approaches for their immunomodulatory and anti-inflammatory properties, which are encoded in their secretome including extracellular vesicles (EVs). The Hoffa fat pad (HFP) located infrapatellarly harbours MSCs that could assist in tissue homeostasis in osteoarthritic joints. Intraarticular injection therapies based on blood products could modulate the populations of released HFP-MSC-EVs in a quantitative manner. METHODS: To obtain amounts of HFP-MSC-derived EVs that allow pre-clinical evaluation, suitable EV production systems need to be developed. This work investigates the release of EVs from primary HFP-MSCs cultivated in a 3D environment using microcarrier suspension culture in a vertical wheel bioreactor in comparison to conventional 2D culture. To simulate an intraarticular blood product therapy, cultures were treated with citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) before EV collection. HFP-MSC-EVs are enriched via ultrafiltration and characterised via Western Blot, nanoparticle tracking analysis in scatter as well as fluorescence mode. EV potency was determined via RT-qPCR analysing the expression of type II and X collagen (COL2 and COL10), as well as inducible nitric oxide synthase (iNOS) in primary OA chondrocytes. RESULTS: Blood product supplementation elevated HFP-MSC metabolic activity as determined via XTT assay over the course of 14 days. 3D culture resulted in a roughly 100-fold EV yield compared to 2D culture and elevated number of EVs released per cell. Total protein content correlated with the EV concentration. While typical EV marker proteins such as CD9, CD63 or Alix were detected in total protein extracts, CD9 and CD73 colocalised on individual EVs highlighting their cell origin. The type of blood product treatment did not affect the size or concentration of EVs obtained from HFP-MSCs. Assessing potency of 3D culture EVs in comparison to 2D EVs revealed superior biological activity with regard to inhibition of inflammation, inhibition of chondrocyte hypertrophy and induction of cartilage-specific ECM production. CONCLUSIONS: HFP-MSCs proliferate in presence of human blood products indicating that animal serum in culture media can be avoided in the future. The culture of HFP-MSCs in the employed bioreactor was successfully used to generate quantities of EVs that could allow evaluation of HFP-MSC-EV-mediated effects in pre-clinical settings. In addition, EV potency of 3D EVs is superior to EVs obtained in conventional 2D culture flasks.

Calebin A modulates inflammatory and autophagy signals for the prevention and treatment of osteoarthritis.

Introduction: Osteoarthritis (OA) is associated with excessive cartilage degradation, inflammation, and decreased autophagy. Insufficient efficacy of conventional monotherapies and poor tissue regeneration due to side effects are just some of the unresolved issues. Our previous research has shown that Calebin A (CA), a component of turmeric (Curcuma longa), has pronounced anti-inflammatory and anti-oxidative effects by modulating various cell signaling pathways. Whether CA protects chondrocytes from degradation and apoptosis in the OA environment (EN), particularly via the autophagy signaling pathway, is however completely unclear. Methods: To study the anti-degradative and anti-apoptotic effects of CA in an inflamed joint, an in vitro model of OA-EN was created and treated with antisense oligonucleotides targeting NF-κB (ASO-NF-κB), and IκB kinase (IKK) inhibitor (BMS-345541) or the autophagy inhibitor 3-methyladenine (3-MA) and/or CA to affect chondrocyte proliferation, degradation, apoptosis, and autophagy. The mechanisms underlying the CA effects were investigated by MTT assays, immunofluorescence, transmission electron microscopy, and Western blot analysis in a 3D-OA high-density culture model. Results: In contrast to OA-EN or TNF-α-EN, a treatment with CA protects chondrocytes from stress-induced defects by inhibiting apoptosis, matrix degradation, and signaling pathways associated with inflammation (NF-κB, MMP9) or autophagy-repression (mTOR/PI3K/Akt), while promoting the expression of matrix compounds (collagen II, cartilage specific proteoglycans), transcription factor Sox9, and autophagy-associated proteins (Beclin-1, LC3). However, the preventive properties of CA in OA-EN could be partially abrogated by the autophagy inhibitor 3-MA. Discussion: The present results reveal for the first time that CA is able to ameliorate the progression of OA by modulating autophagy pathway, inhibiting inflammation and apoptosis in chondrocytes, suggesting that CA may be a novel therapeutic compound for OA.

Development of pluripotent stem cell-derived epidermal organoids that generate effective extracellular vesicles in skin regeneration.

Cellular skin substitutes such as epidermal constructs have been developed for various applications, including wound healing and skin regeneration. These cellular models are mostly derived from primary cells such as keratinocytes and fibroblasts in a two-dimensional (2D) state, and further development of three-dimensional (3D) cultured organoids is needed to provide insight into the in vivo epidermal phenotype and physiology. Here, we report the development of epidermal organoids (EpiOs) generated from induced pluripotent stem cells (iPSCs) as a novel epidermal construct and its application as a source of secreted biomolecules recovered by extracellular vesicles (EVs) that can be utilized for cell-free therapy of regenerative medicine. Differentiated iPSC-derived epidermal organoids (iEpiOs) are easily cultured and expanded through multiple organoid passages, while retaining molecular and functional features similar to in vivo epidermis. These mature iEpiOs contain epidermal stem cell populations and retain the ability to further differentiate into other skin compartment lineages, such as hair follicle stem cells. By closely recapitulating the epidermal structure, iEpiOs are expected to provide a more relevant microenvironment to influence cellular processes and therapeutic response. Indeed, iEpiOs can generate high-performance EVs containing high levels of the angiogenic growth factor VEGF and miRNAs predicted to regulate cellular processes such as proliferation, migration, differentiation, and angiogenesis. These EVs contribute to target cell proliferation, migration, and angiogenesis, providing a promising therapeutic tool for in vivo wound healing. Overall, the newly developed iEpiOs strategy as an organoid-based approach provides a powerful model for studying basic and translational skin research and may also lead to future therapeutic applications using iEpiOs-secreted EVs.

Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids.

Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 × 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.

Impact of Fc receptors and host characteristics on myeloid phagocytic response to rituximab-treated 3D-cultured B-cell lymphoma.

Antibody-based immunotherapy is successful in treating cancer, but its effectiveness varies among patients. Therefore, understanding myeloid phagocytic responses to therapeutic antibodies is critical. Immunoglobulin Fc receptors and host characteristics were evaluated in phagocytosis of 3D-cultured CD20+ B-cell lymphoma (spheroids) treated with different anti-CD20 rituximab (RTX) monoclonal antibody isotypes. Monocytes from healthy donors of different ages and sexes were isolated, and their Fc receptors for IgG (FcγRI, FcγRIIa, FcγRIIIa) and IgA (FcαRI) were determined, as well as Fc receptor gene polymorphisms. Antibody-dependent phagocytosis was assessed using flow cytometry, confocal imaging, and Fc receptor blocking. RTX isotypes showed varying efficacy in stimulating the phagocytosis of spheroids. RTX-IgG3 proved to be the most efficient, followed by RTX-IgG1. Monocytes infiltrated RTX-treated spheroids at the periphery but migrated also into the core when stimulated with RTX-IgG3. Blocking FcγRI or FcγRIIa, but not FcγRIIIa, with antibodies inhibited RTX-IgG1 and RTX-IgG3-mediated phagocytosis. Monocytes from younger women demonstrated higher FcγRI and FcγRIIa levels compared to older women, while older men displayed increasing FcγRI and FcγRIIIa levels compared to younger men. Monocytes from younger women displayed greater phagocytic activity compared to older women, while older men had better IgG-mediated phagocytosis than younger men. Single Fc receptor levels, or FcγRIIa and FcγRIIIa genetic variants, had a low correlation with phagocytic intensity, likely as a result of multiple engagements of Fcreceptors for IgG-mediated phagocytosis. In conclusion, antibody isotype, Fc receptors, age, and sex influence tumor phagocytosis. This study exposes the relationship between host traits and the efficacy of therapeutic antibodies, providing insights into cancer immunotherapy treatment.

Three-dimensional culture conditioned bone marrow MSC secretome accelerates wound healing in a burn injury mouse model.

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising regenerative therapeutic approach for wound healing. To determine the effects of cultured MSCs as a 2D monolayer (2D-MSCs) and 3D spheroids (3D-MSCs) on their secretomes, and to examine the effect of 3D-MSC secretomes on endothelial cells (ECs) and MSCs in a burn injury mouse model. MSCs were cultured as 2D monolayers (2D-MSCs) and 3D spheroids (3D-MSCs) and their cellular characteristics were evaluated by western blotting. 2D-MSC and 3D-MSC secretomes (condition medium: CM) were analyzed using an angiogenic array. The activation of ECs by 2D-MSC and 3D-MSC CMs was examined in cellular proliferation, migration, and tube formation assays. The wound healing effects of 2D-MSCs and 3D-MSCs were determined in vivo using a burn injury mouse model. 3D culture conditions altered the markers of components that regulate cell survival, cytoskeletal, adhesion, and proliferation. Interleukin-6 (IL-6), vascular endothelial growth factor A (VEGFA), IL-8, and chemokine (CXC motif) ligand 1 (CXCL1) were present at high levels in the CM of 3D-MSCs compared with 2D-MCs. 3D-MSC-CMs promoted the proliferation, migration, and tube formation of ECs. Furthermore, 3D-MSC treatment enhanced wound healing in a burn injury mouse model. 3D culture improves proangiogenic factors in the MSC secretome and 3D-MSCs represent a new cell-based treatment strategy for wound healing.

In vitro spermatogenesis: In search of fully defined conditions.

A complete reconstruction of spermatogenesis in vitro under fully defined conditions still has not been achieved. However, many techniques have been proposed to get closer to that aim. Here we review the current progress in the field. At first, we describe the most successful technique, the organ culture method, which allows to produce functional haploid cells. However, this method is based on the culturing of intact testis tissue with unknown factors acting inside it. Then we discuss different types of 3D-cultures where specific testicular cell populations may be aggregated and the impact of each cell population may be examined. Unfortunately, germ cell development does not proceed further than the pachytene stage of meiosis there, with rare exceptions. Finally, we describe recent studies that focus on germ cells in a conventional adherent cell culture. Such studies thoroughly examine issues with in vitro meiosis and provide insight into the mechanisms of meiotic initiation.

Modelling Alzheimer's disease using human brain organoids: current progress and challenges.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by gradual memory loss and declining cognitive and executive functions. AD is the most common cause of dementia, affecting more than 50 million people worldwide, and is a major health concern in society. Despite decades of research, the cause of AD is not well understood and there is no effective curative treatment so far. Therefore, there is an urgent need to increase understanding of AD pathophysiology in the hope of developing a much-needed cure. Dissecting the cellular and molecular mechanisms of AD pathogenesis has been challenging as the most commonly used model systems such as transgenic animals and two-dimensional neuronal culture do not fully recapitulate the pathological hallmarks of AD. The recent advent of three-dimensional human brain organoids confers unique opportunities to study AD in a humanised model system by encapsulating many aspects of AD pathology. In the present review, we summarise the studies of AD using human brain organoids that recapitulate the major pathological components of AD including amyloid-ß and tau aggregation, neuroinflammation, mitochondrial dysfunction, oxidative stress and synaptic and circuitry dysregulation. Additionally, the current challenges and future directions of the brain organoids modelling system are discussed.

Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations.

Organoids are 3D-culture systems composed of tissue-specific primary cells that self-organize and self-renew, creating structures similar to those of their tissue of origin. Testicular organoids (TOs) may recreate conditions of the testicular niche in domestic and wild cattle; however, no previous TO studies have been reported in the bovine species. Thus, in the present study, we sought to generate and characterize bovine TOs derived from primary testicular cell populations including Leydig, Sertoli and peritubular myoid cells. Testicular cells were isolated from bovine testes and cultured in ultra-low attachment (ULA) plates and Matrigel. TOs were cultured in media supplemented from day 3 with 100 ng/mL of BMP4 and 10 ng/mL of FGF2 and from day 7 with 15 ng/mL of GDNF. Testicular cells were able to generate TOs after 3 days of culture. The cells positive for STAR (Leydig) and COL1A (peritubular myoid) decreased (p < 0.05), whereas cells positive for WT1 (Sertoli) increased (p < 0.05) in TOs during a 28-day culture period. The levels of testosterone in media increased (p < 0.05) at day 28 of culture. Thus, testicular cells isolated from bovine testes were able to generate TOs under in vitro conditions. These bovine TOs have steroidogenic activity characterized by the production of testosterone.

Culturing of Cardiac Cells in 3D Spheroids Modulates Their Expression Profile and Increases Secretion of Proangiogenic Growth Factors.

We studied the effect of 3D-culturing of cells in the form of cardiospheres on the expression of genes encoding vascular progenitor cell markers and angiogenesis regulators and on the production of proangiogenic factors. Cardiospheres were obtained by culturing mouse cardiac explants followed by self-assembly on poly-D-lysine. Gene expression was assessed by real-time PCR, and the production of proangiogenic factors was assessed by Microarray analysis of the cell secretome. It was found that cells in the cardiospheres in comparison with 2D-culture of cardiosphere-forming cells demonstrated increased expression of vascular progenitor cell markers (Pdgfrα, Kit, and Vegfr1) and angiogenesis regulatory factors (Vegf, Fgf2, and Angpt1), as well as an enhanced secretion of proangiogenic factors (ANGPT1, VEGF, CXCL16, and PIGF-2). Thus, culturing of cells in the form of cardiospheres can be considered as a basis for developing approaches to increasing their angiogenic activity and regenerative properties.

Progress and perspective of organoid technology in cancer-related translational medicine.

Organoids are in vitro simplified and miniature microcosms of internal organs, which have aroused great interest in tissue development, multiple disease models, clinical diagnosis, as well as high-throughput drug screening and personalized medicine and so on. The success of physiology-related organoid culture has greatly advanced the translational medicine research in the field of cancer treatment, which was once troubled by the inconsistency between two-dimensional (2D) cell culture and in vivo studies. Especially in recent years, the success rate of establish an organoid has been greatly improved, and the types of organoids have been gradually enriched. Moreover, the utilizing of some the cutting-edge technologies, including gene editing technology such as CRISPR-Cas9, the scope of application of organoid is broadened. In this review, we discuss the latest progress and applications of organoids, and also outline the potential challenges of organoids for future improvement.

Recapitulating Tumor Microenvironment Using AXTEX-4DTM for Accelerating Cancer Research and Drug Screening.

OBJECTIVE: The formation of three-dimensional spheroid tumor model using the scaffold-based platforms has been demonstrated over many years now. 3D tumor models are generated mainly in non-scalable culture systems, using synthetic and biological scaffolds. Many of these models fail to reflect the complex tumor microenvironment and do not allow long-term monitoring of tumor progression. This has resulted in inconsistent data in drug testing assays during preclinical and clinical studies. METHODS: To overcome these limitations, we have developed 3D tissueoids model by using novel AXTEX-4D platform. RESULTS: Cancer 3D tissueoids demonstrated the basic features of 3D cell culture with rapid attachment, proliferation, and longevity with contiguous cytoskeleton and hypoxic core. This study also demonstrated greater drug resistance in 3D-MCF-7 tissueoids in comparison to 2D monolayer cell culture. CONCLUSION: In conclusion, 3D-tissueoids are more responsive than 2D-cultured cells in simulating important tumor characteristics, anti-apoptotic features, and their resulting drug resistance.

Functional tissue-engineered microtissue formed by self-aggregation of cells for peripheral nerve regeneration.

BACKGROUND: Peripheral nerve injury (PNI) is one of the essential causes of physical disability with a high incidence rate. The traditional tissue engineering strategy, Top-Down strategy, has some limitations. A new tissue-engineered strategy, Bottom-Up strategy (tissue-engineered microtissue strategy), has emerged and made significant research progress in recent years. However, to the best of our knowledge, microtissues are rarely used in neural tissue engineering; thus, we intended to use microtissues to repair PNI. METHODS: We used a low-adhesion cell culture plate to construct adipose-derived mesenchymal stem cells (ASCs) into microtissues in vitro, explored the physicochemical properties and microtissues components, compared the expression of cytokines related to nerve regeneration between microtissues and the same amount of two-dimension (2D)-cultured cells, co-cultured directly microtissues with dorsal root ganglion (DRG) or Schwann cells (SCs) to observe the interaction between them using immunocytochemistry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA). We used grafts constructed by microtissues and polycaprolactone (PCL) nerve conduit to repair sciatic nerve defects in rats. RESULTS: The present study results indicated that compared with the same number of 2D-cultured cells, microtissue could secrete more nerve regeneration related cytokines to promote SCs proliferation and axons growth. Moreover, in the direct co-culture system of microtissue and DRG or SCs, axons of DRG grown in the direction of microtissue, and there seems to be a cytoplasmic exchange between SCs and ASCs around microtissue. Furthermore, microtissues could repair sciatic nerve defects in rat models more effectively than traditional 2D-cultured ASCs. CONCLUSION: Tissue-engineered microtissue is an effective strategy for stem cell culture and therapy in nerve tissue engineering.

Cardiac Organoids and Gastruloids to Study Physio-Pathological Heart Development.

Ethical issues restrict research on human embryos, therefore calling for in vitro models to study human embryonic development including the formation of the first functional organ, the heart. For the last five years, two major models have been under development, namely the human gastruloids and the cardiac organoids. While the first one mainly recapitulates the gastrulation and is still limited to investigate cardiac development, the second one is becoming more and more helpful to mimic a functional beating heart. The review reports and discusses seminal works in the fields of human gastruloids and cardiac organoids. It further describes technologies which improve the formation of cardiac organoids. Finally, we propose some lines of research towards the building of beating mini-hearts in vitro for more relevant functional studies.

Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells.

This study evaluates the impact of physiologically relevant oxygen tensions on the response of HepG2 cells to known inducers and hepatotoxic drugs. We compared transcriptional regulation and CYP1A activity after a 48 h exposure at atmospheric culture conditions (20% O2) with representative periportal (8% O2) and perivenous (3% O2) oxygen tensions. We evaluated cellular responses in 2D and 3D cultures at each oxygen tension in parallel, using monolayers and a paper-based culture platform that supports cells suspended in a collagen-rich environment. Our findings highlight that the toxicity, potency, and mechanism of action of drugs are dependent on both culture format and oxygen tension. HepG2 cells in 3D environments at physiologic oxygen tensions better matched primary human hepatocyte data than HepG2 cells cultured under standard conditions. Despite altered transcriptional regulation with decreasing oxygen tensions, we did not observe the zonation patterns of drug-metabolizing enzymes found in vivo. Our approach demonstrates that oxygen is an important regulator of liver function but it is not the sole regulator. It also highlights the utility of the 3D paper-based culture platform for continued mechanistic studies of microenvironmental influences on cellular responses.

Organotypic Models in Drug Development "Tumor Models and Cancer Systems Biology for the Investigation of Anticancer Drugs and Resistance Development".

The landscape of cancer treatment has improved over the past decades, aiming to reduce systemic toxicity and enhance compatibility with the quality of life of the patient. However, at the therapeutic level, metastatic cancer remains hugely challenging, based on the almost inevitable emergence of therapy resistance. A small subpopulation of cells able to survive drug treatment termed the minimal residual disease may either harbor resistance-associated mutations or be phenotypically resistant, allowing them to regrow and become the dominant population in the therapy-resistant tumor. Characterization of the profile of minimal residual disease represents the key to the identification of resistance drivers that underpin cancer evolution. Therapeutic regimens must, therefore, be dynamic and tailored to take into account the emergence of resistance as tumors evolve within a complex microenvironment in vivo. This requires the adoption of new technologies based on the culture of cancer cells in ways that more accurately reflect the intratumor microenvironment, and their analysis using omics and system-based technologies to enable a new era in the diagnostics, classification, and treatment of many cancer types by applying the concept "from the cell plate to the patient." In this chapter, we will present and discuss 3D model building and use, and provide comprehensive information on new genomic techniques that are increasing our understanding of drug action and the emergence of resistance.

Brain Organoids as Model Systems for Genetic Neurodevelopmental Disorders.

Neurodevelopmental disorders (NDDs) are a group of disorders in which the development of the central nervous system (CNS) is disturbed, resulting in different neurological and neuropsychiatric features, such as impaired motor function, learning, language or non-verbal communication. Frequent comorbidities include epilepsy and movement disorders. Advances in DNA sequencing technologies revealed identifiable genetic causes in an increasingly large proportion of NDDs, highlighting the need of experimental approaches to investigate the defective genes and the molecular pathways implicated in abnormal brain development. However, targeted approaches to investigate specific molecular defects and their implications in human brain dysfunction are prevented by limited access to patient-derived brain tissues. In this context, advances of both stem cell technologies and genome editing strategies during the last decade led to the generation of three-dimensional (3D) in vitro-models of cerebral organoids, holding the potential to recapitulate precise stages of human brain development with the aim of personalized diagnostic and therapeutic approaches. Recent progresses allowed to generate 3D-structures of both neuronal and non-neuronal cell types and develop either whole-brain or region-specific cerebral organoids in order to investigate in vitro key brain developmental processes, such as neuronal cell morphogenesis, migration and connectivity. In this review, we summarized emerging methodological approaches in the field of brain organoid technologies and their application to dissect disease mechanisms underlying an array of pediatric brain developmental disorders, with a particular focus on autism spectrum disorders (ASDs) and epileptic encephalopathies.

Toward Cardiac Regeneration: Combination of Pluripotent Stem Cell-Based Therapies and Bioengineering Strategies.

Cardiovascular diseases represent the major cause of morbidity and mortality worldwide. Multiple studies have been conducted so far in order to develop treatments able to prevent the progression of these pathologies. Despite progress made in the last decade, current therapies are still hampered by poor translation into actual clinical applications. The major drawback of such strategies is represented by the limited regenerative capacity of the cardiac tissue. Indeed, after an ischaemic insult, the formation of fibrotic scar takes place, interfering with mechanical and electrical functions of the heart. Hence, the ability of the heart to recover after ischaemic injury depends on several molecular and cellular pathways, and the imbalance between them results into adverse remodeling, culminating in heart failure. In this complex scenario, a new chapter of regenerative medicine has been opened over the past 20 years with the discovery of induced pluripotent stem cells (iPSCs). These cells share the same characteristic of embryonic stem cells (ESCs), but are generated from patient-specific somatic cells, overcoming the ethical limitations related to ESC use and providing an autologous source of human cells. Similarly to ESCs, iPSCs are able to efficiently differentiate into cardiomyocytes (CMs), and thus hold a real regenerative potential for future clinical applications. However, cell-based therapies are subjected to poor grafting and may cause adverse effects in the failing heart. Thus, over the last years, bioengineering technologies focused their attention on the improvement of both survival and functionality of iPSC-derived CMs. The combination of these two fields of study has burst the development of cell-based three-dimensional (3D) structures and organoids which mimic, more realistically, the in vivo cell behavior. Toward the same path, the possibility to directly induce conversion of fibroblasts into CMs has recently emerged as a promising area for in situ cardiac regeneration. In this review we provide an up-to-date overview of the latest advancements in the application of pluripotent stem cells and tissue-engineering for therapeutically relevant cardiac regenerative approaches, aiming to highlight outcomes, limitations and future perspectives for their clinical translation.

Material-Dependent Formation and Degradation of Bone Matrix-Comparison of Two Cryogels.

Cryogels represent ideal carriers for bone tissue engineering. We recently described the osteogenic potential of cryogels with different protein additives, e.g., platelet-rich plasma (PRP). However, these scaffolds raised concerns as different toxic substances are required for their preparation. Therefore, we developed another gelatin (GEL)-based cryogel. This study aimed to compare the two scaffolds regarding their physical characteristics and their influence on osteogenic and osteoclastic cells. Compared to the PRP scaffolds, GEL scaffolds had both larger pores and thicker walls, resulting in a lower connective density. PRP scaffolds, with crystalized calcium phosphates on the surface, were significantly stiffer but less mineralized than GEL scaffolds with hydroxyapatite incorporated within the matrix. The GEL scaffolds favored adherence and proliferation of the osteogenic SCP-1 and SaOS-2 cells. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin (OPG) levels seemed to be induced by GEL scaffolds. Levels of other osteoblast and osteoclast markers were comparable between the two scaffolds. After 14 days, mineral content and stiffness of the cryogels were increased by SCP-1 and SaOS-2 cells, especially of PRP scaffolds. THP-1 cell-derived osteoclastic cells only reduced mineral content and stiffness of PRP cryogels. In summary, both scaffolds present powerful advantages; however, the possibility to altered mineral content and stiffness may be decisive when it comes to using PRP or GEL scaffolds for bone tissue engineering.