Advances in ex vivo expansion of hematopoietic stem and progenitor cells for clinical applications.

As caretakers of the hematopoietic system, hematopoietic stem cells assure a lifelong supply of differentiated populations that are responsible for critical bodily functions, including oxygen transport, immunological protection and coagulation. Due to the far-reaching influence of the hematopoietic system, hematological disorders typically have a significant impact on the lives of individuals, even becoming fatal. Hematopoietic cell transplantation was the first effective therapeutic avenue to treat such hematological diseases. Since then, key use and manipulation of hematopoietic stem cells for treatments has been aspired to fully take advantage of such an important cell population. Limited knowledge on hematopoietic stem cell behavior has motivated in-depth research into their biology. Efforts were able to uncover their native environment and characteristics during development and adult stages. Several signaling pathways at a cellular level have been mapped, providing insight into their machinery. Important dynamics of hematopoietic stem cell maintenance were begun to be understood with improved comprehension of their metabolism and progressive aging. These advances have provided a solid platform for the development of innovative strategies for the manipulation of hematopoietic stem cells. Specifically, expansion of the hematopoietic stem cell pool has triggered immense interest, gaining momentum. A wide range of approaches have sprouted, leading to a variety of expansion systems, from simpler small molecule-based strategies to complex biomimetic scaffolds. The recent approval of Omisirge, the first expanded hematopoietic stem and progenitor cell product, whose expansion platform is one of the earliest, is predictive of further successes that might arise soon. In order to guarantee the quality of these ex vivo manipulated cells, robust assays that measure cell function or potency need to be developed. Whether targeting hematopoietic engraftment, immunological differentiation potential or malignancy clearance, hematopoietic stem cells and their derivatives need efficient scaling of their therapeutic potency. In this review, we comprehensively view hematopoietic stem cells as therapeutic assets, going from fundamental to translational.

Secretome of bone marrow mesenchymal stromal cells cultured in a dynamic system induces neuroprotection and modulates microglial responsiveness in an α-synuclein overexpression rat model.

BACKGROUND AIMS: Parkinson's disease (PD) is the second most common neurodegenerative disorder. The etiology of the disease remains largely unknown, but evidence have suggested that the overexpression and aggregation of alpha-synuclein (α-syn) play key roles in the pathogenesis and progression of PD. Mesenchymal stromal cells (MSCs) have been earning attention in this field, mainly due to their paracrine capacity. The bioactive molecules secreted by MSCs, i.e. their secretome, have been associated with enhanced neuronal survival as well as a strong modulatory capacity of the microenvironments where the disease develops. The selection of the appropriate animal model is crucial in studies of efficacy assessment. Given the involvement of α-syn in the pathogenesis of PD, the evidence generated from the use of animal models that develop a pathologic phenotype due to the action of this protein is extremely valuable. Therefore, in this work, we established an animal model based on the viral vector-mediated overexpression of A53T α-syn and studied the impact of the secretome of bone marrow mesenchymal stromal cells MSC(M) as a therapeutic strategy. METHODS: Adult male rats were subjected to α-syn over expression in the nigrostriatal pathway to model dopaminergic neurodegeneration. The impact of locally administered secretome treatment from MSC(M) was studied. Motor impairments were assessed throughout the study coupled with whole-region (striatum and substantia nigra) confocal microscopy evaluation of histopathological changes associated with dopaminergic neurodegeneration and glial cell reactivity. RESULTS: Ten weeks after lesion induction, the animals received secretome injections in the substantia nigra pars compacta (SNpc) and striatum (STR). The secretome used was produced from bone marrow mesenchymal stromal cells MSC(M) expanded in a spinner flask (SP) system. Nine weeks later, animals that received the viral vector containing the gene for A53T α-syn and treated with vehicle (Neurobasal-A medium) presented dopaminergic cell loss in the SNpc and denervation in the STR. The treatment with secretome significantly reduced the levels of α-syn in the SNpc and protected the dopaminergic neurons (DAn) within the SNpc and STR. CONCLUSIONS: Our results are aligned with previous studies in both α-syn Caenorhabditis elegans models, as well as 6-OHDA rodent model, revealing that secretome exerted a neuroprotective effect. Moreover, these effects were associated with a modulation of microglial reactivity supporting an immunomodulatory role for the factors contained within the secretome. This further supports the development of new studies exploring the effects and the mechanism of action of secretome from MSC(M) against α-syn-induced neurotoxicity.

Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system.

BACKGROUND & AIMS: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. METHODS: MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. RESULTS: Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). CONCLUSIONS: Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.

Evaluation of the theranostic potential of [64Cu]CuCl2 in glioblastoma spheroids.

BACKGROUND: Glioblastoma is an extremely aggressive malignant tumor with a very poor prognosis. Due to the increased proliferation rate of glioblastoma, there is the development of hypoxic regions, characterized by an increased concentration of copper (Cu). Considering this, 64Cu has attracted attention as a possible theranostic radionuclide for glioblastoma. In particular, [64Cu]CuCl2 accumulates in glioblastoma, being considered a suitable agent for positron emission tomography. Here, we explore further the theranostic potential of [64Cu]CuCl2, by studying its therapeutic effects in advanced three-dimensional glioblastoma cellular models. First, we established spheroids from three glioblastoma (T98G, U373, and U87) and a non-tumoral astrocytic cell line. Then, we evaluated the therapeutic responses of spheroids to [64Cu]CuCl2 exposure by analyzing spheroids' growth, viability, and cells' proliferative capacity. Afterward, we studied possible mechanisms responsible for the therapeutic outcomes, including the uptake of 64Cu, the expression levels of a copper transporter (CTR1), the presence of a cancer stem cell population, and the production of reactive oxygen species (ROS). RESULTS: Results revealed that [64Cu]CuCl2 is able to significantly reduce spheroids' growth and viability, while also affecting cells' proliferation capacity. The uptake of 64Cu, the presence of cancer stem-like cells and the production of ROS were in accordance with the therapeutic response. However, expression levels of CTR1 were not in agreement with uptake levels, revealing that other mechanisms could be involved in the uptake of 64Cu. CONCLUSIONS: Overall, our results further support [64Cu]CuCl2 potential as a theranostic agent for glioblastoma, unveiling potential mechanisms that could be involved in the therapeutic response.

Developing a Cell-Microcarrier Tissue-Engineered Product for Muscle Repair Using a Bioreactor System.

Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provides a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restoring continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. In this study, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. First, patient-isolated SkMDCs were expanded using planar static culture systems. Second, by using a single-use PBS-MINI Vertical-Wheel® bioreactor, the expanded SkMDCs were combined with biocompatible and biodegradable (i.e., directly implantable) poly(lactic-co-glycolic acid) microcarriers prepared by thermally induced phase separation. This process allowed for up to 80% efficiency of SkMDCs to attach to the microcarriers. Importantly, SkMDCs were viable during all the process and maintained their myogenic features (e.g., expression of the CD56 marker) after adhesion and culture on the microcarriers. When SkMDC-containing microcarriers were placed on a culture dish, cells were able to migrate from the microcarriers onto the culture surface and differentiate into multinucleated myotubes, which highlights their potential to regenerate the damaged sphincter muscle after administration into the patient. Overall, this study proposes an innovative method to attach SkMDCs to biodegradable microcarriers, which can provide a new treatment for fecal incontinence.

Bioprocessing Considerations towards the Manufacturing of Therapeutic Skeletal and Smooth Muscle Cells.

Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth muscle, responsible for involuntary and voluntary contraction, respectively. In this review, several features and parameters involved in the bioprocessing of muscle cells are addressed. The cell isolation process is depicted, depending on the type of tissue (smooth or skeletal muscle), followed by the description of the challenges involving the use of adult donor tissue and the strategies to overcome the hurdles of reaching relevant cell numbers towards a clinical application. Specifically, the use of stem/progenitor cells is highlighted as a source for smooth and skeletal muscle cells towards the development of a cellular product able to maintain the target cell's identity and functionality. Moreover, taking into account the need for a robust and cost-effective bioprocess for cell manufacturing, the combination of muscle cells with biomaterials and the need for scale-up envisioning clinical applications are also approached.

Optimized operation of a controlled stirred tank reactor system for the production of mesenchymal stromal cells and their extracellular vesicles.

The therapeutic effects of human mesenchymal stromal cells (MSC) have been attributed mostly to their paracrine activity, exerted through small-secreted extracellular vesicles (EVs) rather than their engraftment into injured tissues. Currently, the production of MSC-derived EVs (MSC-EVs) is performed in laborious static culture systems with limited manufacturing capacity using serum-containing media. In this work, a serum-/xenogeneic-free microcarrier-based culture system was successfully established for bone marrow-derived MSC cultivation and MSC-EV production using a 2  l-scale controlled stirred tank reactor (STR) operated under fed-batch (FB) or fed-batch combined with continuous perfusion (FB/CP). Overall, maximal cell numbers of (3.0 ± 0.12) × 108 and (5.3 ± 0.32) × 108 were attained at Days 8 and 12 for FB and FB/CP cultures, respectively, and MSC(M) expanded under both conditions retained their immunophenotype. MSC-EVs were identified in the conditioned medium collected from all STR cultures by transmission electron microscopy, and EV protein markers were successfully identified by Western blot analysis. Overall, no significant differences were observed between EVs isolated from MSC expanded in STR operated under the two feeding approaches. EV mean sizes of 163 ± 5.27 nm and 162 ± 4.44 nm (p > 0.05) and concentrations of (2.4 ± 0.35) × 1011 EVs/mL and (3.0 ± 0.48) × 1011 EVs/mL (p > 0.05) were estimated by nanoparticle tracking analysis for FB and FB/CP cultures, respectively. The STR-based platform optimized herein represents a major contribution toward the development of human MSC- and MSC-EV-based products as promising therapeutic agents for Regenerative Medicine settings.

Neurodifferentiation and Neuroprotection Potential of Mesenchymal Stromal Cell-Derived Secretome Produced in Different Dynamic Systems.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by the degeneration of the dopamine (DA) neurons in the substantia nigra pars compacta, leading to a loss of DA in the basal ganglia. The presence of aggregates of alpha-synuclein (α-synuclein) is seen as the main contributor to the pathogenesis and progression of PD. Evidence suggests that the secretome of mesenchymal stromal cells (MSC) could be a potential cell-free therapy for PD. However, to accelerate the integration of this therapy in the clinical setting, there is still the need to develop a protocol for the large-scale production of secretome under good manufacturing practices (GMP) guidelines. Bioreactors have the capacity to produce large quantities of secretomes in a scalable manner, surpassing the limitations of planar static culture systems. However, few studies focused on the influence of the culture system used to expand MSC, on the secretome composition. In this work, we studied the capacity of the secretome produced by bone marrow-derived mesenchymal stromal cells (BMSC) expanded in a spinner flask (SP) and in a Vertical-Wheel™ bioreactor (VWBR) system, to induce neurodifferentiation of human neural progenitor cells (hNPCs) and to prevent dopaminergic neuron degeneration caused by the overexpression of α-synuclein in one Caenorhabditis elegans model of PD. Results showed that secretomes from both systems were able to induce neurodifferentiation, though the secretome produced in the SP system had a greater effect. Additionally, in the conditions of our study, only the secretome produced in SP had a neuroprotective potential. Lastly, the secretomes had different profiles regarding the presence and/or specific intensity of different molecules, namely, interleukin (IL)-6, IL-4, matrix metalloproteinase-2 (MMP2), and 3 (MMP3), tumor necrosis factor-beta (TNF-ß), osteopontin, nerve growth factor beta (NGFß), granulocyte colony-stimulating factor (GCSF), heparin-binding (HB) epithelial growth factor (EGF)-like growth factor (HB-EGF), and IL-13. Overall, our results suggest that the culture conditions might have influenced the secretory profiles of cultured cells and, consequently, the observed effects. Additional studies should further explore the effects that different culture systems have on the secretome potential of PD.

Enhanced Proliferative and Osteogenic Potential of Periodontal Ligament Stromal Cells.

Cell-based therapies using periodontal ligament stromal cells (PDLSC) for periodontal regeneration may represent an alternative source for mesenchymal stromal cells (MSC) to MSC derived from bone marrow (MSC(M)) and adipose tissue (MSC(AT)). We aimed to characterize the osteogenic/periodontal potential of PDLSC in comparison to MSC(M) and MSC(AT). PDLSC were obtained from surgically extracted healthy human third molars, while MSC(M) and MSC(AT) were obtained from a previously established cell bank. Flow cytometry, immunocytochemistry, and cell proliferation analyses provided cellular characteristics from each group. Cells from the three groups presented MSC-like morphology, MSC-related marker expression, and multilineage differentiation capacity (adipogenic, chondrogenic, and osteogenic). In this study, PDLSC expressed osteopontin, osteocalcin, and asporin, while MSC(M) and MSC(AT) did not. Of note, only PDLSC expressed CD146, a marker previously applied to identify PDLSC, and presented higher proliferative potential compared to MSC(M) and MSC(AT). Upon osteogenic induction, PDLSC exhibited higher calcium content and enhanced upregulation of osteogenic/periodontal genes compared to MSC(M) and MSC(AT), such as Runx2, Col1A1 and CEMP-1. However, the alkaline phosphatase activity of PDLSC did not increase. Our findings suggest that PDLSC might be a promising cell source for periodontal regeneration, presenting enhanced proliferative and osteogenic potential compared to MSC(M) and MSC(AT).

Bioengineered Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles as an Improved Drug Delivery System: Methods and Applications.

Extracellular vesicles (EVs) are cell-derived nano-sized lipid membranous structures that modulate cell-cell communication by transporting a variety of biologically active cellular components. The potential of EVs in delivering functional cargos to targeted cells, their capacity to cross biological barriers, as well as their high modification flexibility, make them promising drug delivery vehicles for cell-free therapies. Mesenchymal stromal cells (MSCs) are known for their great paracrine trophic activity, which is largely sustained by the secretion of EVs. MSC-derived EVs (MSC-EVs) retain important features of the parental cells and can be bioengineered to improve their therapeutic payload and target specificity, demonstrating increased therapeutic potential in numerous pre-clinical animal models, including in the treatment of cancer and several degenerative diseases. Here, we review the fundamentals of EV biology and the bioengineering strategies currently available to maximize the therapeutic value of EVs, focusing on their cargo and surface manipulation. Then, a comprehensive overview of the methods and applications of bioengineered MSC-EVs is presented, while discussing the technical hurdles yet to be addressed before their clinical translation as therapeutic agents.

Hypothermic Preservation of Adipose-Derived Mesenchymal Stromal Cells as a Viable Solution for the Storage and Distribution of Cell Therapy Products.

Cell and gene therapies (CGT) have reached new therapeutic targets but have noticeably high prices. Solutions to reduce production costs might be found in CGT storage and transportation since they typically involve cryopreservation, which is a heavily burdened process. Encapsulation at hypothermic temperatures (e.g., 2-8 °C) could be a feasible alternative. Adipose tissue-derived mesenchymal stromal cells (MSC(AT)) expanded using fetal bovine serum (FBS)- (MSC-FBS) or human platelet lysate (HPL)-supplemented mediums (MSC-HPL) were encapsulated in alginate beads for 30 min, 5 days, and 12 days. After bead release, cell recovery and viability were determined to assess encapsulation performance. MSC identity was verified by flow cytometry, and a set of assays was performed to evaluate functionality. MSC(AT) were able to survive encapsulated for a standard transportation period of 5 days, with recovery values of 56 ± 5% for MSC-FBS and 77 ± 6% for MSC-HPL (which is a negligible drop compared to earlier timepoints). Importantly, MSC function did not suffer from encapsulation, with recovered cells showing robust differentiation potential, expression of immunomodulatory molecules, and hematopoietic support capacity. MSC(AT) encapsulation was proven possible for a remarkable 12 day period. There is currently no solution to completely replace cryopreservation in CGT logistics and supply chain, although encapsulation has shown potential to act as a serious competitor.

From Promise to Reality: Bioengineering Strategies to Enhance the Therapeutic Potential of Extracellular Vesicles.

Extracellular vesicles (EVs) have been the focus of great attention over the last decade, considering their promising application as next-generation therapeutics. EVs have emerged as relevant mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. Given their natural ability to shuttle messages between cells, EVs have been explored both as inherent therapeutics in regenerative medicine and as drug delivery vehicles targeting multiple diseases. However, bioengineering strategies are required to harness the full potential of EVs for therapeutic use. For that purpose, a good understanding of EV biology, from their biogenesis to the way they are able to shuttle messages and establish interactions with recipient cells, is needed. Here, we review the current state-of-the-art on EV biology, complemented by representative examples of EVs roles in several pathophysiological processes, as well as the intrinsic therapeutic properties of EVs and paradigmatic strategies to produce and develop engineered EVs as next-generation drug delivery systems.

Impact of Donor Age on the Osteogenic Supportive Capacity of Mesenchymal Stromal Cell-Derived Extracellular Matrix.

Mesenchymal stromal cells (MSC) have been proposed as an emerging cell-based therapeutic option for regenerative medicine applications as these cells can promote tissue and organ repair. In particular, MSC have been applied for the treatment of bone fractures. However, the healing capacity of these fractures is often compromised by patient's age. Therefore, considering the use of autologous MSC, we evaluated the impact of donor age on the osteogenic potential of bone marrow (BM)-derived MSC. MSC from older patients (60 and 80 years old) demonstrated impaired proliferative and osteogenic capacities compared to MSC isolated from younger patients (30 and 45 years old), suggesting that aging potentially changes the quantity and quality of MSC. Moreover, in this study, we investigated the capacity of the microenvironment [i.e., extracellular matrix (ECM)] to rescue the impaired proliferative and osteogenic potential of aged MSC. In this context, we aimed to understand if BM MSC features could be modulated by exposure to an ECM derived from cells obtained from young or old donors. When aged MSC were cultured on decellularized ECM derived from young MSC, their in vitro proliferative and osteogenic capacities were enhanced, which did not happen when cultured on old ECM. Our results suggest that the microenvironment, specifically the ECM, plays a crucial role in the quality (assessed in terms of osteogenic differentiation capacity) and quantity of MSC. Specifically, the aging of ECM is determinant of osteogenic differentiation of MSC. In fact, old MSC maintained on a young ECM produced higher amounts of extracellularly deposited calcium (9.10 ± 0.22 vs. 4.69 ± 1.41 µg.µl-1.10-7 cells for young ECM and old ECM, respectively) and up-regulated the expression of osteogenic gene markers such as Runx2 and OPN. Cell rejuvenation by exposure to a functional ECM might be a valuable clinical strategy to overcome the age-related decline in the osteogenic potential of MSC by recapitulating a younger microenvironment, attenuating the effects of aging on the stem cell niche. Overall, this study provides new insights on the osteogenic potential of MSC during aging and opens new possibilities for developing clinical strategies for elderly patients with limited bone formation capacity who currently lack effective treatments.

Mesenchymal Stromal Cells (MSCs): A Promising Tool for Cell-Based Angiogenic Therapy.

The Mesenchymal stromal cells (MSCs) are a diverse subset of adult multipotent precursors, known for their potential therapeutic properties in regenerative medicine mainly sustained by paracrine effects through secretion of a variety of biologically active molecules. MSC secretome includes a wide range of soluble protein factors, composed of growth factors and cytokines, and vesicular components, which transfer proteins and genetic material modulating the host microenvironment. In particular, MSC-derived secretome mediates the different steps of the angiogenic process, inducing endothelial cell functions in vitro and promoting angiogenesis in vivo. As a result, MSCs have been widely explored as a promising cell-based therapy in diseases caused by insufficient angiogenesis. Numerous studies of myocardial infarction, ischemic stroke, and critical limb ischemia in animals have shown that human MSCs can enhance angiogenesis and accelerate tissue regeneration. This extensive preclinical work encouraged the study of these remarkable cells for the treatment of these disorders in human clinical settings. The present review provides a comprehensive overview of the pro-angiogenic potential of MSCs and paracrine effectors of their secretome. In addition, bioengineering strategies, including ex vivo preconditioning and genetic modification approaches, to enhance MSC innate angiogenic properties, and thereby therapeutic potency, will be presented. Finally, an update on completed preclinical and clinical studies with MSCs for the treatment of ischemia-related diseases will be discussed.

Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34+-enriched cells.

BACKGROUND: Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo. METHODS: UCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed. RESULTS: MSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells. CONCLUSIONS: AT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.

Bone Matrix Non-Collagenous Proteins in Tissue Engineering: Creating New Bone by Mimicking the Extracellular Matrix.

Engineering biomaterials that mimic the extracellular matrix (ECM) of bone is of significant importance since most of the outstanding properties of the bone are due to matrix constitution. Bone ECM is composed of a mineral part comprising hydroxyapatite and of an organic part of primarily collagen with the rest consisting on non-collagenous proteins. Collagen has already been described as critical for bone tissue regeneration; however, little is known about the potential effect of non-collagenous proteins on osteogenic differentiation, even though these proteins were identified some decades ago. Aiming to engineer new bone tissue, peptide-incorporated biomimetic materials have been developed, presenting improved biomaterial performance. These promising results led to ongoing research focused on incorporating non-collagenous proteins from bone matrix to enhance the properties of the scaffolds namely in what concerns cell migration, proliferation, and differentiation, with the ultimate goal of designing novel strategies that mimic the native bone ECM for bone tissue engineering applications. Overall, this review will provide an overview of the several non-collagenous proteins present in bone ECM, their functionality and their recent applications in the bone tissue (including dental) engineering field.

Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues.

BACKGROUND: Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors. The overexpression of VEGF combined with the intrinsic properties of MSC could represent a promising strategy towards angiogenic therapies. METHODS: We established a microporation-based protocol to transfect human MSC using VEGF-encoding MC (MC-VEGF). VEGF production levels were measured by an enzyme-linked immunosorbent assay and a quantitative polymerase chain reaction. The in vitro angiogenic potential of transfected cells was quantified using cell tube formation and migration functional studies. RESULTS: MSC isolated from BM, AT or UCM showed similar levels of VEGF secretion after transfection with MC-VEGF. Those values were significantly higher when compared to non-transfected cells, indicating an effective enhancement of VEGF production. Transfected cells displayed higher in vitro angiogenic potential than non-transfected controls, as demonstrated by functional in vitro assays. No significant differences were observed among cells from different sources. CONCLUSIONS: Minicircles can be successfully used to transiently overexpress VEGF in human MSC, regardless of the cell tissue source, representing an important advantage in a clinical context (i.e., angiogenic therapy) because a standard protocol might be applied to MSC of different tissue sources, which can be differentially selected according to the application (e.g., autologous versus allogeneic settings).

Dimethyloxalylglycine, a small molecule, synergistically increases the homing and angiogenic properties of human mesenchymal stromal cells when cultured as 3D spheroids.

Strategies aiming at increasing the survival and paracrine activity of human mesenchymal stromal cells (MSCs) are of utmost importance to achieve the full therapeutic potential of these cells. Herein, we propose both physical and biochemical strategies to enhance the survival, homing, angiogenic, and immunomodulatory properties of MSCs in vitro. To that purpose, we compared the effect of exposing either 2D monolayer or 3D spheroids of MSCs to (i) hypoxia (2% O2 ) or to (ii) a hypoxic-mimetic small molecule, dimethyloxalylglycine (DMOG), with cells cultured at 21% O2 . 3D-cultured MSC spheroids evidenced higher survival upon exposure to oxidative stress and expressed higher levels of factors involved in tissue repair processes, namely tumor necrosis factor-stimulated gene-6, matrix metalloproteinase-2, and vascular endothelial growth factor. MSCs cultured as 3D spheroids and further exposed to hypoxia or hypoxic-mimetic conditions provided by DMOG synergistically favored the expression of the cell surface marker C-X-C chemokine receptor type-4, involved in homing processes to injured tissues, and adhesion to extracellular matrix components as fibronectin. These results highlight the role of ex vivo preconditioning approaches, presenting a novel strategy that combine biochemical stimuli with 3D spheroid organization of MSCs to maximize their tissue regeneration potential.

Copper-64 Chloride Exhibits Therapeutic Potential in Three-Dimensional Cellular Models of Prostate Cancer.

Prostate cancer (PCa) is the second most common cancer type in men, and in advanced metastatic stages is considerable incurable. This justifies the need for efficient early diagnostic methods and novel therapies, particularly radiopharmaceuticals with the potential for simultaneous diagnosis and therapy (theranostics). We have previously demonstrated, using monolayer-cultured cells, that copper-64 chloride, a promising theranostic agent for PCa, has the potential to induce significant damage in cancer cells while having minimal side effects in healthy tissues. Here, we further explored this compound for its theranostic applications using more advanced PCa cellular models, specifically multicellular spheroids. Namely, we evaluated the cellular uptake of 64CuCl2 in three human PCa spheroids (derived from 22RV1, DU145, and LNCaP cells), and characterized the growth profile and viability of those spheroids as well as the clonogenic capacity of spheroid-derived cells after exposure to 64CuCl2. Furthermore, the populations of cancer stem cells (CSCs), known to be important for cancer resistance and recurrence, present in the spheroid models were also evaluated using two different markers (CD44 and CD117). 64CuCl2 was found to have significant detrimental effects in spheroids and spheroid-derived cells, being able to reduce their growth and impair the viability and reproductive ability of spheroids from both castration-resistant (22RV1 and DU145) and hormone-naïve PCa (LNCaP). Interestingly, resistance to 64CuCl2 treatment seemed to be related with the presence of a CSC population, since the most resistant spheroids, derived from the DU145 cell line, had the highest initial percentage of CSCs among the three cell lines under study. Altogether, these results clearly highlight the theranostic potential of 64CuCl2.

Corrigendum: Successful Use of Human AB Serum to Support the Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cell in a Microcarrier-Based Platform.

[This corrects the article DOI: 10.3389/fbioe.2020.00307.].