Large‐scale expansion of pre‐isolated bone marrow mesenchymal stromal cells in serum‐free conditions

The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum‐free media (SFM) for their ability to support the growth and expansion of pre‐isolated undifferentiated bone marrow‐derived MSCs (BM‐MSCs) and compared the results with cells grown in standard FBS‐containing medium as control. In addition, based on initial screening results, BD Mosaic? Mesenchymal Stem Cell Serum‐free (BD‐SFM) medium was evaluated in large‐scale cultures for the performance and culture characteristics of BM‐MSCs. Of the five different serum‐free media, BD‐SFM enhanced BM‐MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS‐10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC‐specific markers. Significant differences were observed between BD‐SFM and control medium in terms of population doublings (PDs), cell yield, CFU‐F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD‐SFM‐cultured MSCs were also found to retain the differentiation potential, immune‐privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD‐SFM supports large‐scale expansion of BM‐MSCs for therapeutic use. Copyright © 2014 John Wiley & Sons, Ltd.     

Optimized human platelet lysate as novel basis for a serum‐, xeno‐, and additive‐free corneal endothelial cell and tissue culture

The expansion of donor‐derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno‐free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice‐grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3‐fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1‐mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno‐free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy.     

Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow‐derived mesenchymal stem cells

Human adult bone marrow‐derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi‐lineage differentiation potential. We compare the different basal culture media DMEM‐F12, DMEM‐high glucose (DMEM‐HG), DMEM‐low glucose (DMEM‐LG), knock‐out DMEM (DMEM‐KO) and Mesencult^® on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO‐SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up‐scaling to provide sufficient numbers for transplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Osteogenic capacity of human BM‐MSCs,AT‐MSCs and their co‐cultures using HUVECs in FBS and PL supplemented media

Human bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and human adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) are the most frequently used stem cells in tissue engineering. Due to major clinical demands, it is necessary to find an optimally safe and efficient way for large‐scale expansion of these cells. Considering the nutritional source in the culture medium and method, this study aimed to analyze the effects of FBS‐ and PL‐supplemented media on osteogenesis in stem cell mono‐ and co‐cultures with human umbilical vein endothelial cells (HUVECs). Results showed that cell metabolic activity and proliferation increased in PL‐ compared to FBS‐supplemented media in mono‐ and co‐cultures for both BM‐MSCs and AT‐MSCs. In addition, calcium deposition was cell type dependent and decreased for BM‐MSCs but increased for AT‐MSCs in PL‐supplemented medium in both mono‐ and co‐cultures. Based on the effects of co‐cultures, BM‐MSCs/HUVECs enhanced osteogenesis compared to BM‐MSCs monocultures in both FBS‐ and PL‐supplemented media whereas AT‐MSCs/HUVECs showed similar results compared to AT‐MSCs monocultures. Copyright © 2013 John Wiley & Sons, Ltd.     

Synergic effects of VEGF‐A and SDF‐1 on the angiogenic properties of endothelial progenitor cells

Here we investigated the impact of hypoxic environment on the angiogenic properties of early‐outgrowth endothelial progenitor cells (EPCs), with particular focus on the role of secreted vascular endothelial growth factor‐A (VEGF‐A) and stromal derived factor‐1 (SDF‐1) in mediating these effects. We found that cultured EPCs secreted factors with paracrine effects on chemotaxis, migration, proliferation and tube formation of mature endothelial cells (ECs), and these properties were not affected by hypoxia. Depletion of VEGF‐A did not change the ability of EPC‐conditioned medium (CM) to promote EC migration and tube formation in vitro, suggesting that the pro‐angiogenic paracrine effects of EPCs did not totally rely on the presence of VEGF‐A. These findings were confirmed by in vivo experiments, on a mouse model of hind limb ischaemia, which showed that VEGF‐depleted EPC‐CM sustained tissue perfusion at the same level as complete EPC‐CM. However, concomitant deletion of VEGF‐A and SDF‐1 in EPC‐CM impaired the pro‐angiogenic properties of EPC‐CM, by inhibition of EC spreading in culture, tube‐like structure formation on Matrigel support, in vivo neovessels formation and ischaemic hind limb regeneration. Taken together, our data demonstrate that: (i) hypoxia does not affect the capacity of EPCs to support the angiogenic process; (ii) the absence of either VEGF‐A or SDF‐1 from EPC‐CM can be rescued by the presence of the other one, so that the overall angiogenic effects remain unchanged; and (iii) and the concomitant deletion of VEGF‐A and SDF‐1 from EPC‐CM impairs its pro‐angiogenic effect, both in vitro and in vivo. Copyright © 2016 John Wiley & Sons, Ltd.     

Iron dose‐dependent differentiation and enucleation of human erythroblasts in serum‐free medium

Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron‐saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum‐free media may be highly dependent on the concentration of iron. Here adult human CD34⁺ cells were cultured in a serum‐free medium containing dosed levels of iron‐saturated transferrin (holo‐Tf, 0.1–1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo‐Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo‐Tf dose increased. These data suggest that adequate holo‐Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum‐free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA.     

Efficient cryopreservation of human mesenchymal stem cells using silkworm hemolymph‐derived proteins

Cryopreservation methods for human mesenchymal stem cells (hMSCs) typically depend on the presence of fetal bovine serum (FBS) with dimethyl sulphoxide (DMSO), which is not appropriate for therapeutic applications. In our previous study, we found that storage protein 2 (SP2), a natural material derived from silkworm hemolymph, has an inhibitory effect on the generation of reactive oxygen species (ROS). In this study, we used SP2 as an alternative to establish an effective, low‐DMSO and FBS‐free cryopreservation agent for the cryostorage of hMSCs. We investigated the cell viability and stem cell characterization of umbilical cord‐derived MSCs in different freezing media through the freezing and thawing process. We also evaluated the efficacy of cryostorage using these media over 1 week and 1 year. When the cell characteristics (cell viability and stemness) were analysed after thawing, those obtained using 5 mg/ml SP2 were comparable to those obtained using a freezing medium with FBS. The stable cell viability and characteristics were shown even after 1 year of cryopreservation. In addition, when the cells were differentiated into adipocytes and osteocytes, we confirmed that the differentiation behaviours of the thawed cells were well maintained. The positive results could be also obtained when SP2 was applied to other MSCs. The results clearly indicate that SP2 could be used as an alternative to FBS for a freezing medium with reduced DMSO. Copyright © 2016 John Wiley & Sons, Ltd.     

Cell culture of differentiated human salivary epithelial cells in a serum‐free and scalable suspension system: The salivary functional units model

Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease‐modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum‐free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub‐optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10 days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell‐specific markers.     

Endothelial invasive response in a co‐culture model with physically‐induced osteodifferentiation

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co‐culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity‐induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co‐culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co‐culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen‐like morphology, suggesting that cell‐cell interaction in the co‐culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co‐culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre‐vascularized bone grafts. Copyright © 2012 John Wiley & Sons, Ltd.     

Challenging the great vascular wall: Can we envision a simple yet comprehensive therapy for stroke?

Stroke is a leading cause of death in adult life, closely behind ischemic heart disease, and causes a significant and abiding socioeconomic burden. However, current therapies are not able to ensure full neurologic and/or sequelae‐free recovery to all stroke survivors. We believe treatment efficacy and patient rehabilitation could be enhanced significantly by targeting blood‐brain barrier (BBB) deregulation and inflammation‐induced barrier loss that occurs after stroke. In this pathological context, bone marrow‐derived endothelial progenitor cells (EPC) enter the bloodstream towards the lesion site, but their insufficient numbers and impaired angiogenic ability compromise neurovascular regeneration. In this context, cell‐based therapies have become increasingly appealing since treating patients with large numbers of mesenchymal or hematopoietic stem/progenitor cells alone may boost repair. However, this approach could be met with several challenges in terms of logistics and cost; hence, the development of a drug delivery system suitable for intravenous administration and functionalized for selective uptake by circulating EPC could enhance their restorative potential without perceived complications. The ability to encapsulate proangiogenic and anti‐inflammatory agents, such as retinoic acid, and to safely and easily deliver them systemically may open new therapeutic perspectives for the treatment of cerebrovascular disorders. Copyright © 2017 John Wiley & Sons, Ltd.     

Fibroblast growth factor improves the motility of human mesenchymal stem cells expanded in a human plasma‐derived xeno‐free medium through αVβ3 integrin

Human mesenchymal stem cells (MSC) are being explored for cell therapies targeting varied human diseases. For that, cells are being expanded in vitro, many times with fetal bovine serum (FBS) as the main source of growth factors. However, animal‐derived components should not be used, to avoid immune rejection from the patient that receives the MSC. To solve this issue, different xeno‐free media are being developed, and an industrial‐grade human plasma fraction (SCC) is a promising candidate to substitute FBS. Indeed, we have previously shown that MSC expanded in SCC‐medium maintain their phenotype and genetic stability. However, a reduction on MSC motility was observed when comparing with MSC motility on FBS‐medium. Thus, in this present study, we have tested different factors to improve the motility of MSC in SCC‐medium. Time lapse assays and experiments with transwells revealed that supplementation of the xeno‐free medium with FGF or PDGF, but not TNF‐α or SDF‐1, increased MSC motility. Interestingly, FGF and PDGF supplementation also led to alterations on MSC morphology to a shape similar to the one observed when using FBS. The mechanism behind the effect of FGF on MSC motility involved the increased expression of αVβ3 integrin. Furthermore, assays with small molecule inhibitors revealed that the signalling molecule p38 MAPK is important for MSC motility and that MEK/ERK and PI3K/AKT also have a role on FGF‐supplemented expanded MSC. Thus, it was found that FGF supplementation can improve the motility of xeno‐free‐expanded MSC and that the cells motility is regulated by αVβ3 integrin.     

Optimization of the use of a pharmaceutical grade xeno‐free medium for in vitro expansion of human mesenchymal stem/stromal cells

Human bone marrow‐derived mesenchymal stem/stromal cells (hMSCs) are considered promising therapeutic agents in the field of cell therapy and regenerative medicine, mainly due to their relative facility to be isolated, multi‐differentiation potential, and immunomodulatory role. However, their application in clinics requires a crucial step of in vitro expansion. Most of the protocols for hMSCs in vitro culture use foetal bovine serum as medium supplement that, being from animal origin, presents several safety concerns and may initiate xenogeneic immune responses after cells transplantation. This work reports the optimization of a pharmaceutical‐grade xeno‐free strategy for hMSCs in vitro expansion based on the supplementation of basal medium with a pharmaceutical‐grade human plasma‐derived supplement for cell culture (SCC) and 2 human growth factors (bFGF and TGFβ1), plus a coating of human plasma fibronectin (Fn). After 4 weeks in culture, this strategy improves hMSCs expansion yield about 4.3‐fold in comparison with foetal bovine serum supplementation and 4.5‐fold compared with a commercially available xeno‐free medium. hMSCs expanded in SCC‐based formulation maintained their phenotype and differentiation capacity into osteogenic, adipogenic, and chondrogenic lineages, without alterations in cell karyotype. Overall, the SCC‐based medium appears to be an excellent alternative for the xeno‐free expansion of hMSCs as therapeutic agents for clinical applications.     

Defining human endothelial progenitor cells

Summary.  There is no specific marker to identify an endothelial progenitor cell (EPC) and this deficiency is restricting the ability of an entire field of research in defining these cells. We will review current methods to define EPC in the human system and suggest approaches to define better the cell populations involved in neoangiogenesis. PubMed was used to identify articles via the search term 'endothelial progenitor cell' and those articles focused on defining the term were evaluated. The only human cells expressing the characteristics of an EPC, as originally proposed, are endothelial colony forming cells. A variety of hematopoietic cells including stem and progenitors, participate in initiating and modulating neoangiogenesis. Future studies must focus on defining the specific hematopoietic subsets that are involved in activating, recruiting, and remodeling the vascular networks formed by the endothelial colony forming cells.     

Granulocyte colony‐stimulating factor (G‐CSF) depresses angiogenesis in vivo and in vitro: implications for sourcing cells for vascular regeneration therapy

Summary. Background: The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G‐CSF administration. Objective: To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G‐CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Patients/Methods: Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G‐CSF‐mobilized samples were collected before administration, at apheresis harvest, and at follow‐up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony‐forming unit (CFU)‐EPC and EOC assays. The effect of exogenous G‐CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Results: Our data show that G‐CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU‐EPCs. Furthermore, G‐CSF administration did not mobilize CD34+CD133? cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G‐CSF inhibited CFU‐EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. Conclusions: G‐CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G‐CSF may come only from a paracrine effect from HPCs.     

Traumatized muscle‐derived multipotent progenitor cells recruit endothelial cells through vascular endothelial growth factor‐A action

Traumatized muscle, such as that debrided from blast injury sites, is considered a promising and convenient tissue source for multipotent progenitor cells (MPCs), a population of adult mesenchymal stem cell (MSC)‐like cells. The present study aimed to assess the regenerative therapeutic potential of human traumatized muscle‐derived MPCs, e.g., for injury repair in the blast‐traumatized extremity, by comparing their pro‐angiogenic potential in vitro and capillary recruitment activity in vivo to those of MSCs isolated from human bone marrow, a widely‐used tissue source. MPCs were tested for their direct and indirect effects on human microvascular endothelial cells (ECs) in vitro. The findings reported here showed that MPC‐conditioned culture medium (MPC‐CM), like MSC‐CM, promoted EC‐cord network branching. Silent (si)RNA‐mediated silencing of vascular endothelial growth factor‐A (VEGF‐A) expression in MPCs attenuated this effect. In a chick embryonic chorioallantoic membrane in vivo angiogenesis assay, MPCs encapsulated in photocrosslinked gelatin scaffold recruited blood vessels more efficiently than either MSCs or human foreskin fibroblasts. Together, these findings support the potential application of traumatized muscle‐derived MPCs in cell‐based regenerative medicine therapies as a result of their influence on EC organization. Copyright © 2017 John Wiley & Sons, Ltd.     

Transient serum exposure regimes to support dual differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (MSCs), which can generate both osteoblasts and chondrocytes, represent an ideal resource for orthopaedic repair using tissue‐engineering approaches. One major difficulty for the development of osteochondral constructs using undifferentiated MSCs is that serum is typically used in culture protocols to promote differentiation of the osteogenic component, whereas existing chondrogenic differentiation protocols rely on the use of serum‐free conditions. In order to define conditions which could be compatible with both chondrogenic and osteogenic differentiation in a single bioreactor, we have analysed the efficiency of new biphasic differentiation regimes based on transient serum exposure followed by serum‐free treatment. MSC differentiation was assessed either in serum‐free medium or with a range of transient exposure to serum, and compared to continuous serum‐containing treatment. Although osteogenic differentation was not supported in the complete absence of serum, marker expression and extensive mineralization analyses established that 5 days of transient exposure triggered a level of differentiation comparable to that observed when serum was present throughout. This initial phase of serum exposure was further shown to support the successful chondrogenic differentiation of MSCs, comparable to controls maintained in serum‐free conditions throughout. This study indicates that a culture based on temporal serum exposure followed by serum‐free treatment is compatible with both osteogenic and chondrogenic differentiation of MSCs. These results will allow the development of novel strategies for osteochondral tissue engineering approaches using MSCs for regenerative medicine. Copyright © 2012 John Wiley & Sons, Ltd.     

Human endothelial colony‐forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold‐based tissue engineering

One of the major challenges in tissue engineering is to supply larger three‐dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)‐derived endothelial colony‐forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue‐culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo‐expanded ECFCs are a novel, very potent cell source for scaffold‐based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Whatever their differentiation status,human progenitor derived – or mature – endothelial cells induce osteoblastic differentiation of bone marrow stromal cells

Association of the bone‐forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co‐culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular‐like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co‐cultured with human endothelial progenitor cells in order to assess whether progenitor‐derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co‐culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non‐orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co‐cultures were undertaken: HBMSCs–PDECs, HBMSCs–HUVECs, HOPs–PDECs, HOPs–HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co‐culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real‐time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono‐ and co‐cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Interactive endothelial phenotype maintenance and osteogenic differentiation of adipose tissue stromal vascular fraction SSEA‐4+‐derived cells

Bone formation relies on complex processes that require well‐orchestrated interactions between several cell types, such as bone‐forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co‐culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co‐culture of microvascular‐like ECs and pre‐OBs, both derived from the SSEA‐4⁺ cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA‐4⁺ hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre‐OBs and maintenance of the EC phenotype. Co‐cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre‐OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31⁺ cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre‐OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co‐culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA‐4⁺ hASCs as a single‐cell source is promising to attain 3D bone‐like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Hydrogel surfaces to promote attachment and spreading of endothelial progenitor cells

Endothelialization of artificial vascular grafts is a challenging process in cardiovascular tissue engineering. Functionalized biomaterials could be promising candidates to promote endothelialization in repair of cardiovascular injuries. The purpose of this study was to synthesize hyaluronic acid (HA) and heparin‐based hydrogels that could promote adhesion and spreading of endothelial progenitor cells (EPCs). We report that the addition of heparin into HA‐based hydrogels provides an attractive surface for EPCs promoting spreading and the formation of an endothelial monolayer on the hydrogel surface. To increase EPC adhesion and spreading, we covalently immobilized CD34 antibody (Ab) on HA–heparin hydrogels, using standard EDC/NHS amine‐coupling strategies. We found that EPC adhesion and spreading on CD34 Ab‐immobilized HA–heparin hydrogels was significantly higher than their non‐modified analogues. Once adhered, EPCs spread and formed an endothelial layer on both non‐modified and CD34 Ab‐modified HA–heparin hydrogels after 3 days of culture. We did not observe significant adhesion and spreading when heparin was not included in the control hydrogels. In addition to EPCs, we also used human umbilical cord vein endothelial cells (HUVECs), which adhered and spread on HA–heparin hydrogels. Macrophages exhibited significantly less adhesion compared to EPCs on the same hydrogels. This composite material could possibly be used to develop surface coatings for artificial cardiovascular implants, due to its specificity for EPC and endothelial cells on an otherwise non‐thrombogenic surface. Copyright © 2012 John Wiley & Sons, Ltd.