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《Cytotherapy》2014,16(10):1371-1383
Background aimsThe purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo.MethodsCultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media.ResultsIsolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects.ConclusionsL-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism. 相似文献
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Shahnaz Razavi Mohamad Reza Razavi Mahsa Kheirollahi-Kouhestani Mohammad Mardani Fatemeh Sadat Mostafavi 《Biochemical and biophysical research communications》2013
Adipose-derived stem cells (ADSCs) and bone marrow stem cells (BMSCs) can be equally proper in the treatment of neurodegenerative diseases. However, ADSCs have practical benefits. In this study, we attempted to induce the secretion of neurotrophic factors (NTF) in human ADSCs. We then evaluated the effects of co-culture with NTF secreting cells in neural differentiation of human ADSCs. Isolated human ADSCs were induced to neurotrophic factors secreting cells. To evaluate the in vitro effects of NTF-secreting ADSCs on neurogenic differentiation of ADSCs, we used neurogenic induction medium (control group), the combination of neurogenic medium and conditioned medium, or co-cultured NTF-secreting ADSCs which were encapsulated in alginate beads (co-culture) for 7 days. ELISA showed increased (by about 5 times) release of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in NTF-secreting ADSCs compared to human ADSCs. Real time RT-PCR analysis revealed that NTF-secreting ADSCs highly expressed NGF and BDNF. In addition, co-culture with NTF-secreting ADSCs could also promote neuronal differentiation relative to gliogenesis. Overall, NTF-secreting ADSCs secrete a range of growth factors whose levels in culture could promote neuronal differentiation and could support the survival and regeneration in a variety of neurodegenerative diseases. 相似文献
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Osteogenic cells differentiated from bone marrow-derived mesenchymal stromal cells (MSC) hold much promise in bone tissue engineering and reconstructive surgery. There is a dire need for well-defined and efficient protocols to promote the osteogenesis of ex vivo cultured MSC. Hence, this study investigated whether a combination of chemical stimuli (ascorbic acid, beta-glycerophosphate and dexamethasone) and culture media conditioned by a human foetal osteoblast cell line (hFOB) had any synergistic effect on the osteogenesis of MSC. Conditioned media with or without prior heat shock treatment (42 degrees C for 1 h) of the hFOB cell line, were collected and tested on rabbit MSC cultures, in the presence and absence of chemical stimuli. Osteogenic differentiation of MSC was assessed on both day 14 and 21 of ex vivo culture. The results showed conclusively that conditioned media promoted osteogenesis of MSC, which was further enhanced by prior heat shock-treatment of the hFOB cells, as well as by the presence of chemical stimuli. Among all experimental groups, the combination of culture medium conditioned by heat shocked hFOB cells together with chemical stimuli, exhibited the highest level of calcium mineralization, as assessed by Von Kossa staining. This provides clear evidence of a synergistic effect of conditioned media, heat shock and chemical stimuli. It is hoped that the data may contribute to the development of a more well-defined and efficient in vitro culture protocol to promote the osteogenesis of MSC for both clinical and non-clinical applications. 相似文献
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BACKGROUND: The multipotency of stromal cells has been studied extensively. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into cells of multilineage. Different methods and reagents have been used to induce the differentiation of MSC. We investigated the efficacy of different growth factors in inducing MSC differentiation into neurons. METHODS: MSC from human BM were isolated and cultured in media supplemented with 10% FBS. These cells were identified and later induced to differentiate into neuron-like cells using different neurotrophic factors. Three different growth factors were used, either alone or in combination: brain-derived neurotrophic factor, epidermal growth factor and neural growth factor. RESULTS: After 10 days of culture, MSC showed neuron-like morphologic changes. Immunostaining showed that these cells expressed markers for neurons (growth-associated protein-43, neuron-specific nuclear protein and neurofilament 200 kDa) and expression of these markers suggested the transition of immature stages to more mature stages of neuron-like cells. DISCUSSION: Our results show that BM-derived MSC can differentiate not only into target cells of mesodermal origin but also neuron-like cells of ectodermal origin. The findings show that a combination of growth factors is more effective in inducing MSC into neuron-like cells. 相似文献
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《European journal of cell biology》2022,101(4):151270
BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge.ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells.MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM).ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield.ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments. 相似文献
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Lushaj EB Anstadt E Haworth R Roenneburg D Kim J Hematti P Kohmoto T 《Cytotherapy》2011,13(4):400-406
Background aimsFor many years the human heart has been considered a terminally differentiated organ with no regenerative potential after injury. Recent studies, however, have cast doubt on this long-standing dogma. The objective of this study was to investigate the presence of and characterize mesenchymal stromal cells (MSC) in the adult mouse heart. The impact of MSC on growth and differentiation of adult cardiac stem cells (CSC) was also analyzed.MethodsA combination of lineage-negative/c-kit-negative (Lin?/c-kit?) immunoselection with a plastic-adhesion technique was used to isolate cardiac-derived MSC. The differentiation capacity and expression of surface markers were analyzed. To investigate the impact of MSC on growth and differentiation of adult CSC, Green Fluorescent Protein (GFP+) adult CSC were co-cultured with GFP? cardiac-derived MSCResultsMSC were present in the adult mouse heart and they met the criteria established to define mouse MSC. They expressed surface markers and were able to differentiate, in a controlled manner, into multiple lineages. In addition, cardiac-derived MSC promoted the survival and expansion of adult CSC in vitroConclusionsMSC can be isolated from the mouse heart and they promote growth and differentiation of adult CSC. The findings from this study could have a significant beneficial impact on future heart failure treatment. Co-culture and co-implantation of cardiac-derived MSC with adult CSC could provide extensive cardiac regeneration and maintenance of the CSC population after implanted into the heart. 相似文献
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Battula VL Bareiss PM Treml S Conrad S Albert I Hojak S Abele H Schewe B Just L Skutella T Bühring HJ 《Differentiation; research in biological diversity》2007,75(4):279-291
Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation. 相似文献
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Antonio Casado-Díaz Carlos Ferreiro-Vera Feliciano Priego-Capote Gabriel Dorado María Dolores Luque-de-Castro José Manuel Quesada-Gómez 《Genes & nutrition》2014,9(1)
Metabolites derived from the polyunsaturated fatty acids (PUFA) may modulate the mesenchymal stromal cell (MSC) differentiation. Such cells can differentiate into different cellular types, including adipocytes and osteoblasts. Aging favors the bone marrow MSC differentiation toward the former, causing a loss of bone density associated with pathologies like osteoporosis. The omega-6 arachidonic acid (AA) favors MSC adipogenesis to a greater extent than omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we study the joint action of both PUFA. Thus, not induced and induced to adipocyte or osteoblast MSC were treated with 20 μM of each PUFA (either AA, AA + DHA or AA + EPA). The expression of osteogenic and adipogenic molecular markers, the alox15b lipoxygenase gene expression and the 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acids (HETE) derived from the AA metabolism in the culture media were determined. The results show that the adipogenesis induction of AA is not suppressed by the joint presence of EPA and DHA. In fact, both increased the adipogenic effect of AA on MSC differentiated into osteoblasts. The different HETE concentrations increased in cultures supplemented with AA, albeit such concentrations were lower in the cultures induced to differentiate, mainly at day 21 after the induction. Furthermore, the reduction in the HETE concentration was correlated with a higher expression of the alox15b gene. These results highlight the PUFA metabolism differences between uninduced and induced MSC to differentiate into adipocytes and osteoblasts, besides the relevant role of the lipoxygenase gene expression in adipogenesis induction. 相似文献
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Hong-Tian Zhang Zhi-Liang Liu Xue-Qin Yao Zhi-Jun Yang Ru-Xiang Xu 《Cytotherapy》2012,14(10):1203-1214
Background aimsThe characteristics, such as morphologic and phenotypic characteristics and neural transdifferentiation ability, of mesenchymal stromal cells (MSC) derived from different origins have yet to be reported for cases isolated from the same individual.MethodsThe proliferation capacity, secretion ability of neurotrophins (NT) and neural differentiation ability in rat MSC isolated from bone marrow (BMSC) and adipose tissue (ADSC) were compared from the same animal.ResultsThe ADSC had a significantly higher proliferation capacity than BMSC according to cell cycle and cumulative population doubling analyses. The proportion of cells expressing neural markers was greater in differentiated ADSC than in differentiated BMSC. Furthermore, the single neurosphere derived from ADSC showed stronger expansion and differentiation abilities than that derived from BMSC. The findings from Western blot lent further support to the immunocytochemical data. The mRNA and protein levels of nerve growth factor (NGF) and brain-derived growth factor (BDNF) expressed in ADSC were significantly higher than those in BMSC at different stages before and following induction.ConclusionsOur study suggests that the proliferation ability of ADSC is superior to that of BMSC. Furthermore, differentiated ADSC expressed higher percentages of neural markers. As one possible alternative source of BMSC, ADSC may have wide potential for treating central nervous system (CNS) diseases. 相似文献
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Erdal Karaoz Selda Ayhan Gülçin Gacar Ayça Aksoy Gökhan Duruksu Alparslan Okçu P. Çetinalp Demircan A. Eker Sariboyaci Figen Kaymaz Murat Kasap 《Cytotherapy》2010,12(3):288-302
Background aimsStem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat.MethodsImmunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied.ResultsWe found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells.ConclusionsThe immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes. 相似文献
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Background aimsThe ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood.MethodsC57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU.ResultsAt a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105+). A 3-fold reduction in the percentage of CD105+ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105+ cells coincided with a 10–20% increase in the frequency of proliferating CD105? cells. Removal of CD105? stroma caused increased proliferation in CD105+ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105+ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105? cells.ConclusionsThis work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC. 相似文献
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《Cytotherapy》2019,21(9):987-1003
Background aimsTissue engineering technology is a promising therapeutic strategy in peripheral nerve injury. Schwann cells (SCs) are deemed to be a vital component of cell-based nerve regeneration therapies. Many methods for producing SC-like cells derived from adipose-derived stromal cells (ADSCs) have been explored, but their phenotypic and functional characteristics remain unsatisfactory.MethodsWe investigated whether human ADSCs can be induced to differentiate into mature and stable SC-like cells with the addition of insulin, progestero``ne and glucocorticoids. The phenotypic and functional characteristics of new differentiated ADSCs (modified SC-like cells) were evaluated by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytochemistry in vitro. Cells loaded into collagen sponge biomaterials were implanted around transected sciatic nerves with a 10-mm gap in vivo. The axon regrowth and functional recovery of the regenerated nerves were assessed by immunohistochemistry and Walking footprint analysis.ResultsAfter differentiation induction, the modified SC-like cells showed significantly up-regulated levels of S100B and P0 and enhanced proliferative and migratory capacities. In addition, the modified SC-like cells showed increased secretion of neurotrophic factors, and their functional characteristics were maintained for more than 3 weeks after removing the induction reagents. The modified SC-like cells exhibited significantly enhanced axon regrowth, myelination and functional recovery after sciatic nerve injury.ConclusionsOverall, the results suggest that this modified induction method can induce human ADSCs to differentiate into cells with the molecular and functional properties of mature SCs and increase the promotion of peripheral nerve regeneration. 相似文献
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Ryutaro Shohara Akihito Yamamoto Sachiko Takikawa Akira Iwase Hideharu Hibi Fumitaka Kikkawa Minoru Ueda 《Cytotherapy》2012,14(10):1171-1181
Background aimsMesenchymal stromal cells (MSC) can be isolated from the perivascular connective tissue of umbilical cords, called Wharton's jelly. These human umbilical cord perivascular cells (HUCPVC) might provide therapeutic benefits when treating skeletal or cutaneous malformations in neonatal patients.MethodsHUCPVC were isolated, and their proliferation rate, marker expression and multilineage differentiation potential determined. HUCPVC or their conditioned medium (HUCPVC-CM) was injected into the excisional wound of a mouse splinted-wound model. The effects of the treatment on wound closure were examined by morphohistochemical and gene expression analyses.ResultsHUCPVC expressed typical MSC markers and could differentiate into osteoblastic and adipogenic lineages. HUCPVC transplanted into the mouse wound accelerated wound closure. Immunohistologic analysis showed that the HUCPVC accelerated wound healing by enhancing collagen deposition and angiogenesis via paracrine mechanisms. Furthermore, treatment with HUCPVC-CM alone significantly enhanced wound closure. HUCPVC-CM increased the number of anti-inflammatory M2 macrophages expressing resistin-like molecule (RELM)-α/CD11b and promoted neovessel maturation. Quantitative polymerase chain reaction (PCR) analysis showed that HUCPVC-CM increased the expression of tissue-repairing cytokines interleukin (IL)-10, transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF)-1 and angiopoietin-1 at the healing wound.ConclusionsOur results show that HUCPVC promotes wound healing via multifaceted paracrine mechanisms. Together with their ability to differentiate into the osteogenic linage, HUCPVC may provide significant therapeutic benefits for treating wounds in neonatal patients. 相似文献
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Internalization of nanopolymeric tracers does not alter characteristics of placental cells
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Paolo Bigini Elisa R. Zanier Silvia Saragozza Simona Maciotta Pietro Romele Patrizia Bonassi Signoroni Antonietta Silini Francesca Pischiutta Eliana Sammali Claudia Balducci Martina B. Violatto Laura Talamini David Garry Davide Moscatelli Raffaele Ferrari Mario Salmona Maria Grazia De Simoni Federico Maggi Giuseppe Simoni Francesca Romana Grati Ornella Parolini 《Journal of cellular and molecular medicine》2016,20(6):1036-1048
In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA‐NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV‐MSC). We report that PMMP‐NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP‐NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP‐NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS‐treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP‐NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP‐NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV‐MSC in preclinical models of inflammatory‐driven diseases. 相似文献
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Malini Fonseka Rajesh Ramasamy Boon Chong Tan Heng Fong Seow 《Cell biology international》2012,36(9):793-801
hUCB‐MSC (human umbilical cord blood‐derived mesenchymal stem cells) offer an attractive alternative to bone marrow‐derived MSC for cell‐based therapy by being less invasive a source of biological material. We have evaluated the effect of hUCB‐MSC on the proliferation of K562 (an erythromyeloblastoid cell line) and the cytokine secretion pattern of hUCB‐MSC. Co‐culturing of hUCB‐MSC and K562 resulted in inhibition of proliferation of K562 in a dose‐dependent manner. However, the anti‐proliferative effect was reduced in transwells, suggesting the importance of direct cell‐to‐cell contact. hUCB‐MSC inhibited proliferation of K562, arresting them in the G0/G1 phase. NO (nitric oxide) was not involved in the hUCB‐MSC‐mediated tumour suppression. The presence of IL‐6 (interleukin 6) and IL‐8 were obvious in the hUCB‐MSC conditioned media, but no significant increase was found in 29 other cytokines. Th1 cytokines, IFNα (interferon α), Th2 cytokine IL‐4 and Th17 cytokine, IL‐17 were not secreted by hUCB‐MSC. There was an increase in the number of hUCB‐MSC expressing the latent membrane‐bound form of TGFβ1 co‐cultured with K562. The anti‐proliferative effect of hUCB‐MSC was due to arrest of the growth of K562 in the G0/G1 phase. The mechanisms underlying increased IL‐6 and IL‐8 secretion and LAP (latency‐associated peptide; TGFβ1) by hUCB‐MSC remains unknown. 相似文献