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.     

Three‐dimensional co‐culture of human hepatocytes and mesenchymal stem cells: improved functionality in long‐term bioreactor cultures

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short‐term viability in two‐dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co‐cultured with human bone marrow mesenchymal stem cells (BM‐MSCs) as spheroids in automated, computer‐controlled, stirred‐tank bioreactors with perfusion operation mode. A dual‐step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM‐MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co‐cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated‐dose toxicity testing. Moreover, the excretory activity was maintained in co‐cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM‐MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Local delivery of allogeneic bone marrow and adipose tissue‐derived mesenchymal stromal cells for cutaneous wound healing in a porcine model

Wound healing remains a major challenge in modern medicine. Bone marrow‐ (BM) and adipose tissue‐ (AT) derived mesenchymal stromal/stem cells (MSCs) are of great interest for tissue reconstruction due to their unique immunological properties and regenerative potential. The purpose of this study was to characterize BM and AT‐MSCs and evaluate their effect when administered in a porcine wound model. MSCs were derived from male Göttingen Minipigs and characterized according to established criteria. Allogeneic BM‐ or AT‐MSCs were administered intradermally (1 x 10⁶ cells) into partial‐thickness wounds created on female animals, and covered with Vaseline® gauze or fibrin in a randomized pattern. Animals were euthanized at 7, 10, 14 and 21 days. Tissues were analyzed visually for healing and by microscopic examination for epidermal development and remodelling. Polymerase chain reaction (PCR) was used to detect the presence of male DNA in the specimens. All wounds were healed by 14 days. MSC‐injected wounds were associated with improved appearance and faster re‐epithelialization compared to saline controls. Evaluation of rete ridge depth and architecture showed that MSC treatment promoted a faster rate of epidermal maturation. Male DNA was detected in all samples at days 7 and 10, suggesting the presence of MSCs. We showed the safety, feasibility and potential efficacy of local injection of allogeneic BM‐ and AT‐MSCs for treatment of wounds in a preclinical model. Our data in this large animal model support the potential use of BM‐ and AT‐MSC for treatment of cutaneous wounds through modulation of healing and epithelialization. Copyright © 2013 John Wiley & Sons, Ltd.     

Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co‐culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs,human induced pluripotent stem cell‐derived MSCs and human embryonic stem cell‐derived MSCs

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co‐culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co‐cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC‐MSCs) and embryonic stem cells (hESC‐MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary‐like structures were successfully formed on CPC in vitro in all four co‐cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co‐cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co‐cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co‐cultured with hUCMSCs, hiPSC‐MSCs and hESC‐MSCs achieved new bone and vessel density similar to hUVECs co‐cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC‐MSCs and hESC‐MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co‐cultures of hUVECs with hUCMSCs, hiPSC‐MSCs, hESC‐MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co‐culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Expansion of adipose tissue mesenchymal stromal progenitors in serum-free medium supplemented with virally inactivated allogeneic human platelet lysate

BACKGROUND: Single‐donor or pooled platelet lysates (PL) can substitute for fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) expansion. However, for clinical applications of MSCs, the use of virally inactivated PL would be desirable. Recently, we have developed a solvent/detergent (S/D)‐treated human PL preparation (S/D‐PL) rich in growth factors. The capacity to use this virally inactivated preparation for MSC expansion needs to be evaluated. STUDY DESIGN AND METHODS: Platelet concentrates were treated by S/D (1% tri‐n‐butyl phosphate and 1% Triton X‐45), extracted by oil, purified by C18 hydrophobic interaction chromatography, and sterile filtered. S/D‐PL was compared to FBS as a medium supplement (10% vol/vol) for isolating, maintaining, and expanding adipose tissue–derived MSCs (AT‐MSCs). Cell morphology; proliferation kinetics; immunophenotype; differentiation capacity toward the chondrogenic, osteogenic, and osteogenic lineages; and cytokine antibody array were assessed. RESULTS: AT‐MSCs had a typical spindle morphology and proliferated in S/D‐PL at least as well as in FBS. Immunophenotype at Passage 7 was characteristic of MSCs and similar for both culture conditions. Differentiation capacity into the three lineages was maintained and chondrogenesis was enhanced by S/D‐PL. In a 120 human cytokine antibody array analysis, 73 cytokines were detected in S/D‐PL, including 22 with a concentration higher than in FBS. CONCLUSION: S/D‐PL is an alternative to FBS for AT‐MSC maintenance and expansion, does not compromise the differentiation capacity nor the immunophenotype, and may accelerate chondrogenesis. S/D‐PL protocols for MSC clinical scale‐up may represent a major step toward challenging new use in stem cell therapies.     

TRANSPLANTATION AND CELLULAR ENGINEERING: Adipose tissue mesenchymal stem cell expansion in animal serum‐free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL (PL 22.9 ± 1.5 hr vs. FBS 106.7 ± 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical‐scale applications mitigating the potential untoward side effects associated with the use of animal‐derived reagents.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Liver extracellular matrix promotes BM‐MSCs hepatic differentiation and reversal of liver fibrosis through activation of integrin pathway

In cell‐based therapies for liver injuries, the clinical outcomes are closely related to the surrounding microenvironment of the transplanted bone marrow mesenchymal stem cells (BM‐MSCs). However, whether liver‐specific ECM (L‐ECM), as one of major microenvironment signals, could regulate the therapeutic effect of BM‐MSCs through changing their biological characteristics is unclear. This study aimed to investigate the hepatogenicity and underlying mechanism of L‐ECM as well as its potential regulative role in the MSC‐based liver recovery. L‐ECM was prepared by homogenization of decellularized whole porcine liver. After three‐dimensional culture with or without the presence of L‐ECM, BM‐MSCs expressed hepatocyte‐specific genes and proteins in an L‐ECM concentration‐dependent manner. Further analysis showed that L‐ECM could activate specific types of integrins (ITGs) as well as their downstream signalling pathways. When the cell/ECM interaction was enhanced by incorporating BM‐MSCs with Mn²⁺, ITGs were activated and the hepatogenic capacity of L‐ECM was improved. The regeneration of rat livers from either acute or chronic fibrosis could also be accelerated after transplantation of Mn²⁺‐treated BM‐MSCs. L‐ECM therefore promotes hepatic differentiation of BM‐MSCs via the ITG pathway and plays a therapeutically beneficial role for stem cell‐based liver regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use

Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future ‘gold standard’ supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue‐specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either ‘young’ or ‘old’ donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of ‘young’ or ‘old’ PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS‐expanded cells. PL‐ and FBS‐expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL‐expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd.     

Electrical stimulation of adipose‐derived mesenchymal stem cells and endothelial cells co‐cultured in a conductive scaffold for potential orthopaedic applications

Electrical stimulation (ES) has emerged as a useful tool to regulate cell behaviour, but the effect of ES on mesenchymal stem cell (MSC)/vasculogenic cell co‐culture has not been investigated. Herein, human adipose‐derived MSCs (AD‐MSCs) and umbilical vein endothelial cells (HUVECs) were co‐cultured in an electrically conductive polypyrrole/chitosan scaffold. Compared with AD‐MSC monoculture, calcium deposition in the co‐culture without and with ES (200 μA for 4 h/day) was 139% and 346% higher, respectively, after 7 days. As the application of ES to AD‐MSC monoculture only increased calcium deposition by 56% compared with that without ES after 7 days, these results indicate that ES and co‐culture with HUVECs have synergistic effects on AD‐MSCs' osteogenic differentiation. ES application also significantly enhanced CD31 expression of HUVECs. In HUVEC monoculture, application of ES increased CD31 expression by 224%, whereas the corresponding increase in AD‐MSC/HUVEC co‐culture with ES application was 62%. The gene expression results indicate that ES enhanced the cellular functions in AD‐MSC and HUVEC monoculture via autocrine bone morphogenetic protein‐2 (BMP‐2) and vascular endothelial growth factor (VEGF), respectively. In co‐culture, crosstalk between AD‐MSCs and HUVECs due to paracrine BMP‐2 and VEGF enhanced the cellular functions compared with the respective monoculture. With application of ES to the AD‐MSC/HUVEC co‐culture, autocrine signalling was enhanced, resulting in further promotion of cellular functions. These findings illustrate that co‐culturing AD‐MSC/HUVEC in a conductive scaffold with ES offers potential benefits for bone defect therapy.     

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.     

Micropatterned three‐dimensional hydrogel system to study human endothelial–mesenchymal stem cell interactions

The creation of vascularized engineered tissues of clinically relevant size is a major challenge of tissue engineering. While it is known that endothelial and mural vascular cells are integral to the formation of stable blood vessels, the specific cell types and optimal conditions for engineered vascular networks are poorly understood. To this end, we investigated the vasculogenic potential of human mesenchymal stem cell (MSC) populations derived from three different sources: (a) bone marrow aspirates; (b) perivascular cells from the umbilical cord vein; and (c) perivascular cells from the umbilical cord artery. Cell populations were isolated and identified as MSCs according to their phenotypes and differentiation potential. Human umbilical vein endothelial cells (HUVECs) were used as a standard for endothelial cells. A novel co‐culture system was developed to study cell–cell interactions in a spatially controlled three‐dimensional (3D) fibrin hydrogel model. Using microfluidic patterning, it was possible to localize hydrogel‐encapsulated HUVECs and MSCs within separate channels spaced at 500, 1000 or 2000 µm. All three MSC populations had similar expression profiles of mesenchymal cell markers and similar capacity for osteogenic and adipogenic differentiation. However, bone marrow‐derived MSCs (but not umbilical vein or artery derived MSCs) showed strong distance‐dependent migration toward HUVECs and supported the formation of stable vascular networks resembling capillary‐like vasculature. The presented approach provides a simple and robust model to study the cell–cell communication of relevance to engineering vascularized tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of serum on the proliferation of bone marrow‐derived mesenchymal stem cells from aged donors and donors with or without chronic heart failure

Many clinical studies of regenerative medicine using bone marrow‐derived mesenchymal stem cells (MSCs) have been conducted globally. We initiated clinical studies using MSCs in 2001 and have now treated over 100 cases with patients aged 0–92 years. In a few cases involving patients with chronic heart failure (CHF), we observed that MSCs proliferated poorly. This contrasts with cell therapy studies wherein MSCs of patients with CHF were used for treatment. The effects of serum on the proliferation of MSCs from donors with normal heart function and with CHF have not been reported. Moreover, whether cell therapy is effective for elderly patients remains uncertain. Therefore, characterization of MSCs from aged donors and/or donors with CHF is urgently required. We retrospectively analysed the population doubling times (PDTs) of MSCs between the first and second passages. Although we had data for many samples of well‐expanded MSCs from aged donors, a positive correlation was observed between donor age and PDT. A trend towards reduced variance in PDTs was observed in MSCs supplemented with fetal bovine serum (FBS) compared with those supplemented with autologous serum. When autologous serum was used, the average PDT of MSCs from donors with CHF was significantly longer than that of MSCs from donors without CHF. In contrast, when FBS was used, similar PDTs were observed in MSCs from donors with and without CHF. Thus, FBS promotes MSC expansion even from donors with CHF and MSC‐based regenerative medicine might be feasible even for elderly patients. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Cultured cell‐derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells

Cell‐derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro‐environment. Therefore, cultured cell‐derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro‐environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell‐derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)‐derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.     

Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells. Based on their properties, several clinical trials have been designed to explore their potential therapeutic effect. Fetal calf serum (FCS, commonly used for in vitro expansion) is an undesirable source of xenogeneic antigens and bears the risk of transmitting contaminations. As an alternative for FCS, platelet lysate (PL) and both autologous and allogeneic human serum have been proposed. The aim of this study is to compare the culture of bone marrow (BM)-derived MSCs in the presence of different serum supplements to determine the effect on cell growth, differentiation potential, and immunologic function.
STUDY DESIGN AND METHODS: MSCs from BM of healthy volunteer donors were grown in the presence of 10% FCS supplemented with 1 ng/mL basic fibroblast growth factor (bFGF), 10% human serum supplemented with 1 ng/mL bFGF, 5% PL, and PL 5% supplemented with 1 ng/mL bFGF (PL plus bFGF).
RESULTS: MSCs that expanded in either medium showed a comparable morphology, phenotype, and proliferative and differentiation capacity. While the presence of MSCs in vitro significantly decreased CD3/CD28-mediated T-cell activation, this effect was significantly higher in MSCs cultured with human serum. Production of interferon-γ was inhibited by cocultured media with MSCs while MSCs also induced a significant inhibition of cell cycle in T cells.
DISCUSSION: In conclusion, PL or autologous serum could offer an alternative to the use of FCS in MSC expansion for clinical use maintaining the same growing potential, phenotype, immunomodulatory properties, and differentiation potential.     

Cell–cell interaction between vocal fold fibroblasts and bone marrow mesenchymal stromal cells in three‐dimensional hyaluronan hydrogel

Mesenchymal stromal cells (MSCs) are multipotential adult cells present in all tissues. Paracrine effects and differentiating ability make MSCs an ideal cell source for tissue regeneration. However, little is known about how interactions between implanted MSCs and native cells influence cellular growth, proliferation, and behaviour. By using an in vitro three‐dimensional (3D) co‐culture assay of normal or scarred human vocal fold fibroblasts (VFFs) and bone marrow‐derived MSCs (BM‐MSCs) in a uniquely suited hyaluronan hydrogel (HyStem–VF), we investigated cell morphology, survival rate, proliferation and protein and gene expression of VFFs and BM‐MSCs. BM‐MSCs inhibited cell proliferation of both normal and scarred VFFs without changes in VFF morphology or viability. BM‐MSCs demonstrated decreased proliferation and survival rate after 7 days of co‐culture with VFFs. Interactions between BM‐MSCs and VFFs led to a significant increase in protein secretion of collagen I and hepatocyte growth factor (HGF) and a decrease of vascular endothelial growth factor (VEGF), monocyte chemotactic protein‐1 (MCP‐1) and interleukin‐6 (IL‐6). In particular, BM‐MSCs significantly upregulated matrix metalloproteinase 1 (MMP1) and HGF gene expression for scarred VFFs compared to normal VFFs, indicating the potential for increases in extracellular matrix remodelling and tissue regeneration. Application of BM‐MSCs‐hydrogels may play a significant role in tissue regeneration, providing a therapeutic approach for vocal fold scarring. Copyright © 2013 John Wiley & Sons, Ltd.     

Placenta-derived MSCs are partially immunogenic and less immunomodulatory than bone marrow-derived MSCs

Over the past few years, mesenchymal stem cells (MSCs) have become of increasing interest for use in the field of regenerative medicine. To date, bone marrow (BM) has been the main source of MSCs (BM-MSCs) for both experimental and clinical studies. However, the use of MSCs derived from BM can be problematic, due to the low number of MSCs found in bone marrow aspirates and the invasive procedure associated with obtaining them. We aimed to develop a method of obtaining high numbers of purified MSCs from placental tissue with minimal expansion and to characterize their phenotype and function relative to BM-MSCs. We show here that placenta-derived MSCs (PD-MSCs) can be isolated with high numbers from whole placental tissue. However, PD-MSCs isolated from whole tissue were often found to be a mixed population of both maternal and neonatal cells. The immunological properties of PD-MSCs and BM-MSCs were compared. PD-MSCs were found to express lower levels of HLA class I and higher levels of PDL-1 and CD1a, compared to BM-MSCs. HLA-DR became upregulated in PD-MSCs following treatment with IFNγ, whereas BM-MSCs expressed constitutively low levels of HLA-DR. Whilst untreated or IFNγ-treated BM-MSCs were incapable of stimulating T cells, we observed a small T cell proliferation in response to the highest concentration of PD-MSCs when treated with IFNγ. It was noted that BM-MSCs were more immunomodulatory than PD-MSCs in this study. We therefore suggest that BM-MSCs may be better candidates for use in commercial regenerative or transplantation medicine.     

Human platelet lysate supports the formation of robust human periodontal ligament cell sheets

The use of stem cell‐derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet‐induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix‐related genes and proteins, such as collagen I, fibronectin and integrin β1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production.