Ex vivo expansion of umbilical cord blood

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.     

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB‐MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper‐exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB‐MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB‐MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM‐MSC) and adipose tissue (AT‐MSC) as reference controls, we observed that CB‐MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10⁹ cells required for cell therapies. CB‐MSC showed karyotype stability after prolonged expansion. Functionally, CB‐MSC could be more readily induced to differentiate into chondrocytes than could BM‐MSC and AT‐MSC. CB‐MSC showed immunosuppressive activity equal to that of BM‐MSC and AT‐MSC. Collectively, our data indicate that viable CB‐MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system. J. Cell. Biochem. 112: 1206–1218, 2011. © 2011 Wiley‐Liss, Inc.     

Proteins secreted by Caco-2 cells support growth of other tumor cells in Chee's Essential Medium without supplements.

The growth promoting effect of several hormones and growth factors on two human colon tumor cell lines (Caco-2 and SW 48) was studied using six different chemically defined serum-free media (SFM). Caco-2 grew in a simple SFM [GF3: Chee's Essential Medium (CEM) plus insulin, transferrin and selenium], whereas, SW 48 cells did not grow in GF3 medium. This suggested that Caco-2 cells probably secrete proteins in SFM which influence attachment and growth of Caco-2 and other tumor cells. Lyophilized Caco-2 conditioned medium and substratum, when added to plain CEM, supported growth of SW 48 and SW 948 cells. The substratum material was more effective than conditioned medium in promoting growth of the cell lines. The substratum material helps attachment and spreading of the cells and, thus, improves growth of the cells over conditioned medium. Caco-2 conditioned medium and substratum were analyzed for their components using SDS-PAGE system and gel filtration chromatography. The substratum was analyzed for the presence of fibronectin and laminin by the ELISA technique. The conditioned medium does not contain TGF alpha and TGF beta. The growth stimulating activity of the conditioned medium is due to a protein component, approximately 58Kd in size.     

Hydroxychloroquine decreases human MSC‐derived osteoblast differentiation and mineralization in vitro

We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favourable effects on BMD. To study the direct effects of HCQ on human MSC‐derived osteoblast activity. Osteoblasts were cultured from human mesenchymal stromal cells (hMSCs). Cultures were treated with different HCQ doses (control, 1 and 5 µg/ml). Alkaline phosphatase activity and calcium measurements were performed to evaluate osteoblast differentiation and activity, respectively. Detailed microarray analysis was performed in 5 µg/ml HCQ‐treated cells and controls followed by qPCR validation. Additional cultures were performed using the cholesterol synthesis inhibitor simvastatin (SIM) to evaluate a potential mechanism of action. We showed that HCQ inhibits both MSC‐derived osteoblast differentiation and mineralization in vitro. Microarray analysis and additional PCR validation revealed a highly significant up‐regulation of the cholesterol biosynthesis, lysosomal and extracellular matrix pathways in the 5 µg/ml HCQ‐treated cells compared to controls. Besides, we demonstrated that 1 µM SIM also decreases MSC‐derived osteoblast differentiation and mineralization compared to controls. It appears that the positive effect of HCQ on BMD cannot be explained by a stimulating effect on the MSC‐derived osteoblast. The discrepancy between high BMD and decreased MSC‐derived osteoblast function due to HCQ treatment might be caused by systemic factors that stimulate bone formation and/or local factors that reduce bone resorption, which is lacking in cell cultures.     

Comparative proteomic profiling of human osteoblast‐derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.     

Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro

The ability of hematopoietic tissue-derived adult stem cells to transdifferentiate into neural progenitor cells offers an interesting alternative to central nervous system (CNS)- or embryonic-derived stem cells as a viable source for cellular therapies applied to brain regeneration. Umbilical cord blood (CB) due to its primitive nature and it unproblematic collection appears as a promising candidate for multipotent stem cell harvest. We developed a negative immunomagnetic selection method that depletes CB from hematopoietic lineage marker-expressing cells, hence isolating a discrete lineage negative (LinNeg) stem cell population (0.1% of CB mononucleated cell [MCN] population). In liquid culture supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand (TPOFLK), CB LinNeg stem cells could expand primitive nonadherent hematopoietic progenitors (up to 47-fold) and simultaneously produce slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks. Laser scanning confocal microscopy analysis identified these adherent cells to express glial fibrillary acidic protein (GFAP). Gene expression analysis showed upregulation of primitive neuroglial progenitor cell markers including, GFAP, nestin, musashi-1, and necdin. ELISA quantification of liquid culture supernatant revealed the in vitro release of transforming growth factor beta-1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF) suggesting their contribution to CB LinNeg stem cell transdifferentiation into neuroglial progenitors. Our study supports that a single CB specimen can be pre-expanded in TPOFLK to produce both primitive hematopoietic and neuropoietic progenitors, hence widening CB clinical potential for cellular therapies.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell‐derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non‐MP secreted factors (Sup) were isolated from serum‐free medium conditioned by human microvascular ECs (HMEC‐1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP‐containing MPs were isolated from cells transduced with CMV‐GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP‐MPs, but not free GFP. Thus, only MP‐associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP‐1, MMP‐3, CCL‐2/MCP‐1 and IL‐6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF‐κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms.     

Mesenchymal stromal cell‐derived extracellular vesicles as cell‐free biologics for the ex vivo expansion of hematopoietic stem cells

Hematopoietic stem cell transplantation (HSCT) is the ultimate choice of treatment for patients with hematological diseases and cancer. The success of HSCT is critically dependent on the number and engraftment efficiency of the transplanted donor hematopoietic stem cells (HSCs). Various studies show that bone marrow‐derived mesenchymal stromal cells (MSCs) support hematopoiesis and also promote ex vivo expansion of HSCs. MSCs exert their therapeutic effect through paracrine activity, partially mediated through extracellular vesicles (EVs). Although the physiological function of EVs is not fully understood, inspiring findings indicate that MSC‐derived EVs can reiterate the hematopoiesis, supporting the ability of MSCs by transferring their cargo containing proteins, lipids, and nucleic acids to the HSCs. The activation state of the MSCs or the signaling mechanism that prevails in them also defines the composition of their EVs, thereby influencing the fate of HSCs. Modulating or preconditioning MSCs to achieve a specific composition of the EV cargo for the ex vivo expansion of HSCs is, therefore, a promising strategy that can overcome several challenges associated with the use of naïve/unprimed MSCs. This review aims to speculate upon the potential role of preconditioned/primed MSC‐derived EVs as “cell‐free biologics,” as a novel strategy for expanding HSCs in vitro.     

Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.     

Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.     

Bone-metastatic prostate carcinoma favors mesenchymal stem cell differentiation toward osteoblasts and reduces their osteoclastogenic potential

Bone homeostasis is achieved by the balance between osteoclast‐dependent bone resorption and osteoblastic events involving differentiation of adult mesenchymal stem cells (MSCs). Prostate carcinoma (PC) cells display the propensity to metastasize to bone marrow where they disrupt bone homeostasis as a result of mixed osteolytic and osteoblastic lesions. The PC‐dependent activation of osteoclasts represents the initial step of tumor engraftment into bone, followed by an accelerated osteoblastic activity and exaggerated bone formation. However, the interactions between PC cells and MSCs and their participation in the disease progression remain as yet unclear. In this study, we show that bone metastatic PC‐3 carcinoma cells release factors that increase the expression by human (h)MSCs of several known pro‐osteoblastic commitment factors, such as α5/β1 integrins, fibronectin, and osteoprotegerin. As a consequence, as shown in an osteogenesis assay, hMSCs treated with conditioned medium (C^edM) derived from PC‐3 cells have an enhanced potential to differentiate into osteoblasts, as compared to hMSCs treated with control medium or with C^edM from non‐metastatic 22RV1 cells. We demonstrate that FGF‐9, one of the factors produced by PC‐3 cells, is involved in this process. Furthermore, we show that PC‐3 C^edM decreases the pro‐osteoclastic activity of hMSCs. Altogether, these findings allow us to propose clues to understand the mechanisms by which PC favors bone synthesis by regulating MSC outcome and properties. J. Cell. Biochem. 112: 3234–3245, 2011. © 2011 Wiley Periodicals, Inc.     

OST1‐mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses

Cold stress is a major environmental factor that negatively affects plant growth and survival. OST1 has been identified as a key protein kinase in plant response to cold stress; however, little is known about the underlying molecular mechanism. In this study, we identified BTF3 and BTF3L (BTF3‐like), β‐subunits of a nascent polypeptide‐associated complex (NAC), as OST1 substrates that positively regulate freezing tolerance. OST1 phosphorylates BTF3 and BTF3L in vitro and in vivo, and facilitates their interaction with C‐repeat‐binding factors (CBFs) to promote CBF stability under cold stress. The phosphorylation of BTF3L at the Ser50 residue by OST1 is required for its function in regulating freezing tolerance. In addition, BTF3 and BTF3L proteins positively regulate the expression of CBF genes. These findings unravel a molecular mechanism by which OST1‐BTF3‐CBF module regulates plant response to cold stress.     

Mesenchymal stem cell–derived conditioned medium attenuate angiotensin II‐induced aortic aneurysm growth by modulating macrophage polarization

Mesenchymal stem cells (MSCs) exhibit therapeutic benefits on aortic aneurysm (AA); however, the molecular mechanisms are not fully understood. The current study aimed to investigate the therapeutic effects and potential mechanisms of murine bone marrow MSC (BM‐MSCs)–derived conditioned medium (MSCs‐CM) on angiotensin II (AngII)‐induced AA in apolipoprotein E‐deficient (apoE^?/?) mice. Murine BM‐MSCs, MSCs‐CM or control medium were intravenously administrated into AngII‐induced AA in apoE^?/? mice. Mice were sacrificed at 2 weeks after injection. BM‐MSCs and MSCs‐CM significantly attenuated matrix metalloproteinase (MMP)‐2 and MMP‐9 expression, aortic elastin degradation and AA growth at the site of AA. These treatments with BM‐MSCs and MSCs‐CM also decreased Ly6c^high monocytes in peripheral blood on day 7 and M1 macrophage infiltration in AA tissues on day 14, whereas they increased M2 macrophages. In addition, BM‐MSCs and MSCs‐CM reduced MCP‐1, IL‐1Ra and IL‐6 expression and increased IL‐10 expression in AA tissues. In vitro, peritoneal macrophages were co‐cultured with BM‐MSCs or fibroblasts as control in a transwell system. The mRNA and protein expression of M2 macrophage markers were evaluated. IL‐6 and IL‐1β were reduced, while IL‐10 was increased in the BM‐MSC systems. The mRNA and protein expression of M2 markers were up‐regulated in the BM‐MSC systems. Furthermore, high concentration of IGF1, VEGF and TGF‐β1 was detected in MSCs‐CM. Our results suggest that MSCs‐CM could prevent AA growth potentially through regulating macrophage polarization. These results may provide a new insight into the mechanisms of BM‐MSCs in the therapy of AA.     

Epirubicin induces apoptosis in osteoblasts through death-receptor and mitochondrial pathways

Epirubicin is an anthracycline and is widely used in tumor treatment, but has toxic and undesirable side effects on wide range of cells and hematopoietic stem cells (HSC). Osteoblasts play important roles in bone development and in supporting HSC differentiation and maturation. It remains unknown whether epirubicin-induced bone loss and hematological toxicity are associated with its effect on osteoblasts. In primary osteoblast cell cultures, epirubicin inhibited cell growth and decreased mineralization. Moreover, epirubicin arrested osteoblasts in the G2/M phase, and this arrest was followed by apoptosis in which both the extrinsic (death receptor-mediated) and intrinsic (mitochondrial-mediated) apoptotic pathways were evoked. The factors involved in the extrinsic apoptotic pathway were increased FasL and FADD as well as activated caspase-8. Those involved in the intrinsic apoptotic pathway were decreased Bcl-2; increased reactive oxygen species, Bax, cytochrome c; and activated caspase-9 and caspase-3. These results demonstrate that epirubicin induced osteoblast apoptosis through the extrinsic and intrinsic apoptotic pathways, leading to the destruction of osteoblasts and consequent lessening of their functions in maintaining bone density and supporting hematopoietic stem cell differentiation and maturation.     

Injection of synthetic mesenchymal stem cell mitigates osteoporosis in rats after ovariectomy

Osteoporosis is a severe skeletal disorder. Patients have a low bone mineral density and bone structural deterioration. Mounting lines of evidence suggest that inappropriate apoptosis of osteoblasts/osteocytes leads to maladaptive bone remodelling in osteoporosis. It has been suggested that transplantation of stem cells, including mesenchymal stem cells, may alter the trajectory of bone remoulding and mitigate osteoporosis in animal models. However, stem cells needed to be carefully stored and characterized before usage. In addition, there is great batch‐to‐batch variation in stem cell production. Here, we fabricated therapeutic polymer microparticles from the secretome and membranes of mesenchymal stem cells (MSCs). These synthetic MSCs contain growth factors secreted by MSCs. In addition, these particles display MSC surface molecules. In vitro, co‐culture with synthetic MSCs increases the viability of osteoblast cells. In a rat model of ovariectomy‐induced osteoporosis, injection of synthetic MSCs mitigated osteoporosis by reducing cell apoptosis and systemic inflammation, but increasing osteoblast numbers. Synthetic MSC offers a promising therapy to manage osteoporosis.     

Insulin-like growth factor binding protein-2 and neurotrophin 3 synergize together to promote the expansion of hematopoietic cells ex vivo

Co-culture of Umbilical Cord Blood (UCB) CD34+ cells with irradiated Mesenchymal Stem Cells (MSCs) without contact increase the expansion of Hematopoietic Progenitor Cells (HPC). Neurotrophin-3 (NT-3) and insulin-like growth factor binding protein-2 (IGFBP-2) are two factors whose expressions were significantly elevated in conditioned media derived from irradiated MSCs. To determine whether these factors are partly responsible for the growth promoting potential of MSCs, we investigated their impact on the growth and differentiation of UCB-CD34+ cells. Addition of either factor alone had little impact on cell growth, however both factors synergized together to increase the expansion of total nucleated cells, erythroids, megakaryocytes (Mk) and CD34+ cells. However, in contrast to MSCs they failed to significantly improve the expansion of hematopoietic progenitors. Consistent with the impact of these factors on hematopoietic cells, both synergized to activate ERK1/2 and AKT in primary human UCB cells. In conclusion, the study demonstrates for the first time that a neurotrophin factor can synergize with IGFBP-2 to promote hematopoietic cell expansion.     

Differential effects of human erythroid burst stimulating activity (HuEBSA) on human cord blood burst forming units-erythroid (BFU-Es) as a function of their differentiation state.

An erythroid stimulating activity which promotes the growth of small bursts probably arising from mature burst forming units-erythroid (BFU-Es) of adult human bone marrow cells and called human erythroid burst stimulating activity (HuEBSA), was previously found in media conditioned by a fetal human kidney cell line. In the present work we report that adding HuEBSA to cultures did not increase the burst number but increased the size of bursts from cord blood (CB) cells. A similar observation was made using stem cell factor (SCF). However, a synergistic effect on the burst number was noted when both HuEBSA and SCF were introduced to cultures. We also noticed that CB erythroid progenitors pre-cultured with 5637-Conditioned Medium [as a source of burst promoting activity (BPA)] and erythopoietin (Epo) for 3 days could be stimulated by HuEBSA but not by SCF. Similar results were obtained when interleukin 3 (IL-3) was introduced with Epo to the pre-cultures. These results suggest that two different populations of erythroid progenitors coexist in cord blood, one is Epo- and IL-3-sensitive, the other solely Epo-sensitive. It also seems probable that HuEBSA acts on erythroid progenitors arising from the more immature erythroid population, since its stimulating activity was evident after a 3-day pre-culture of cord blood cells in Epo and IL-3.     

Serum‐free media for the production of human mesenchymal stromal cells: a review

The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large‐scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)‐supplement for optimal cell proliferation. FBS‐based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum‐use to serum‐free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large‐scale production of MSCs.