The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3⁺/CD19⁺/CD14⁺/CD38⁺‐depleted MNC and CD133⁺‐ or LNGFR⁺‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90⁺/CD73⁺/CD105⁺ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.     

Optimization of primary culture condition for mesenchymal stem cells derived from umbilical cord blood with factorial design

Mesenchymal stem cells (MSCs) can not only support the expansion of hematopoietic stem cells in vitro, but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However, it proved challenging to culture MSCs from umbilical cord blood (UCB) with a success rate of 20–30%. Many cell culture parameters contribute to this outcome and hence optimization of culture conditions is critical to increase the probability of success. In this work, fractional factorial design was applied to study the effect of cell inoculated density, combination and dose of cytokines, and presence of serum and stromal cells. The cultured UCB‐MSC‐like cells were characterized by flow cytometry and their multilineage differentiation potentials were tested. The optimal protocol was identified achieving above 90% successful outcome: 2 × 10⁶ cells/mL mononuclear cells inoculated in Iscove's modified Dulbecco's medium supplied with 10% FBS, 15 ng/mL IL‐3, and 5 ng/mL Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Moreover, the UCB‐MSC‐like cells expressed MSC surface markers of CD13, CD29, CD105, CD166, and CD44 positively, and CD34, CD45, and human leukocyte antigens‐DR (HLA‐DR) negatively. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes, and adipocytes similarly to MSCs derived from bone marrow. In conclusion, we have developed an efficient protocol for the primary culture of UCB‐MSCs by adding suitable cytokines into the culture system. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix

The possibility to isolate canine mesenchymal stem cells (MSCs) from foetal adnexa is interesting since several canine genetic disorders are reported to resemble similar dysfunctions in humans. In this study, we successfully isolated, cytogenetically and molecularly characterized, and followed the differentiation potency of canine MSCs from foetal adnexa, such as amniotic fluid (AF), amniotic membrane (AM), and umbilical cord matrix (UCM). In the three types of cell lines, the morphology of proliferating cells typically appeared fibroblast‐like, and the population doubling time (DT) significantly increased with passage number. For AF‐ and AM‐MSCs, cell viability did not change with passages. In UCM‐MSCs, cell viability remained at approximately constant levels up to P6 and significantly decreased from P7 (P < 0.05). Amnion and UCM‐MSCs expressed embryonic and MSC markers, such as Oct‐4 CD44, CD184, and CD29, whereas AF‐MSCs expressed Oct‐4, CD44. Expression of the hematopoietic markers CD34 and CD45 was not found. Dog leucocyte antigens (DLA‐DRA1 and DLA‐79) were expressed only in AF‐MSCs at P1. Isolated cells of the three cell lines at P3 showed multipotent capacity, and differentiated in vitro into neurocyte, adipocyte, osteocyte, and chondrocyte, as demonstrated by specific stains and expression of molecular markers. Cells at P4 showed normal chromosomal number, structure, and telomerase activity. These results demonstrate that, in dog, MSCs can be successfully isolated from foetal adnexa and grown in vitro. Their proven stemness and chromosomal stability indicated that MSCs could be used as a model to study stem cell biology and have an application in therapeutic programs. Mol. Reprod. Dev. 78:361–373, 2011. © 2011 Wiley‐Liss, Inc.     

Stirred tank bioreactor culture combined with serum‐/xenogeneic‐free culture medium enables an efficient expansion of umbilical cord‐derived mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell‐based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non‐invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)‐free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin‐based Cultispher^®S microcarriers and xeno‐free culture medium for the expansion of umbilical cord matrix (UCM)‐derived MSC. This system enabled the production of 2.4 (±1.1) x10⁵ cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)‐fold increase in cell number. The established protocol was then implemented in a stirred‐tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier‐based stirred culture system, using xeno‐free culture medium that suits the intrinsic features of UCM‐derived MSC represents an important step towards a GMP compliant large‐scale production platform for these promising cell therapy candidates.     

CD45+/CD133+ positive cells expanded from umbilical cord blood expressing PDX‐1 and markers of pluripotency

UCB (human umbilical cord blood) contains cells able to differentiate into non‐haematopoietic cell lineages. It also contains cells similar to primitive ESCs (embryonic stem cells) that can differentiate into pancreatic‐like cells. However, few data have been reported regarding the possibility of expanding these cells or the differential gene expression occurring in vitro. In this study, we expanded formerly frozen UCB cells by treatment with SCF (stem cell factor) and GM‐CSF (granulocyte–macrophage colony stimulating factor) in the presence of VPA (valproic acid). Gene expression profiles for beta cell differentiation and pluripotency (embryo stem cell phenotype) were analysed by RT‐PCR and immunocytochemistry. The results show a dramatic expansion (>150‐fold) of haematopoietic progenitors (CD45⁺/CD133⁺) which also expressed embryo markers of pluripotency (nanog, kfl‐4, sox‐2, oct‐3/4 andc‐myc), nestin, and pancreatic markers such as pax‐4, ngn‐3, pdx‐1 and syt‐1 (that is regulated by pdx‐1 and provides the cells with a Ca⁺⁺ regulation mechanism essential for insulin exocytosis). Our results show that UCB cells can be expanded to produce large numbers of cells of haematopoietic lineage that naturally (without the need of retroviral vectors or transposons) express a gene pattern compatible with endocrine pancreatic precursors and markers of pluripotency. Further investigations are necessary to clarify, first, whether in this context, the embryogenes expressed are functional or not, and secondly, since these cells are safer than cells transfected with retroviral vectors or transposons, whether they would represent a potential tool for clinical application.     

Recruitment of human cord blood-derived endothelial colony-forming cells to sites of tumor angiogenesis

Background aimsEndothelial progenitor cells (EPCs) specifically home to sites of malignant growth, rendering them attractive for anti-cancer therapies. Data are conflicting on the phenotype and quantitative contribution toward tumor angiogenesis based on differing culture assays to outgrow EPCs. To evaluate the origin and early phenotype of EPCs and to define a population with enhanced tumor-targeting capacity, we evaluated a hierarchy of cord blood-derived EPCs modeling the multi-step nature of tumor homing.MethodsCD34⁺ mononuclear cells were isolated from fresh cord blood and cultured to derive endothelial colony-forming cells (ECFCs). Human umbilical vein endothelial cells (HUVECs) served as control. Using intra-vital microscopy, the recruitment was analyzed in mice bearing C6 xenografts. Adhesion, migration, transmigration and differentiation were further addressed.ResultsWithin the primary passage, ECFCs underwent a rapid maturation from a CD45⁺ and CD31⁺ phenotype to a CD45^? and endothelial marker positive phenotype. Assessing in vivo tumor recruitment, ECFCs had the highest activity in all steps analyzed. In vitro, ECFCs demonstrated significantly higher adhesion under static and flow conditions. Similarly, ECFCs exhibited highest migratory and trans-migratory activity toward tumor-conditioned medium. On subcutaneous implantation, only ECFCs formed blood vessels covered with perivascular cells, similar to HUVECs.ConclusionsOur study indicates that ECFCs emerge from a CD45⁺ and CD31⁺ progenitor and rapidly mature in culture. ECFCs have a significantly higher potential for tumor targeting than non-cultured CD34⁺ cells and HUVECs. They are ideal candidates for future cell-based anti-cancer therapies.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

CD45<Superscript>−</Superscript>CD14<Superscript>+</Superscript>CD34<Superscript>+</Superscript> murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term <Emphasis Type="Italic">in vitro</Emphasis> culture

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45⁻CD14⁺CD34⁺ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45⁻CD14⁺ CD34⁺ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured CD34⁺ cells were compared. The average expansion of total cells and CD34⁺ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of CD3⁺ cells declined and the percentage of CD33⁺ cells increased during culture. The engraftment levels of fourteen-day cultured CD34⁺ cells were higher than those of fresh and seven-day cultured CD34⁺ cells. Fourteen-day cultured CD34⁺ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured CD34⁺ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.     

Expanded cryopreserved mesenchymal stromal cells as an optimal source for graft-versus-host disease treatment

Mesenchymal stromal cells (MSC) are fibroblast-like cells present in different types of tissues. Their immunomodulatory potential represents a promising method for post-transplant immunotherapy in the treatment of GVHD (graft-versus-host disease) with suboptimal response to standard immunosuppression. In this study we tested influence of 1–8 month-long cryopreservation on ability of MSC to suppress activation of non-specifically stimulated lymphocytes.We did not observe any changes in proliferation capacity of MSC after thawing. Lymphocytes metabolic activity was inhibited by 30% and number of dividing cells was three times smaller in the presence of MSC. Two activation markers were studied (CD25 and CD69) to confirm preservation of functional cell integrity. Expression of CD25 antigen on CD3⁺CD4⁺ and CD3⁺CD4⁻ cells was decreased in all co-cultivated samples. Level of CD69 expression on CD3⁺CD4⁺ cells was lower in samples with added MSC (10–15% on day +2) but without reaching statistical significance. The lower expression (approximately 5%) was observed also on CD4-cells.The study confirms the preservation of immunomodulatory properties of cryopreserved and re-expanded MSC. Aliquots with cryopreserved cells can represent an optimal source for a quick preparation of MSC cell product with the possibility to apply the same cells repeatedly.     

Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells

The potential of mesenchymal stem cells (MSC) to differentiate into different cell types has opened up the possibility of using these cells clinically to treat a variety of disorders. In this study we describe the use of human umbilical cord blood serum (CBS) as a replacement for fetal bovine serum (FBS) for culturing MSC from different sources. MSC from human and swine bone marrow and human umbilical cord blood were cultured in the presence of DMEM/F12 containing either FBS or CBS. Human MSC cultured in presence of FBS or CBS showed typical fibroblast-like morphology, which is characteristic of MSC. 99% of the cells cultured in FBS had a CD73+/CD105+/CD45- phenotype compared to 96% of cells cultured in CBS. Cells cultured in CBS had a significantly higher cell count as compared to cells cultured in FBS. Swine Bone Marrow MSC cultured in the presence of FBS and CBS were morphologically and phenotypically similar. Human umbilical cord blood serum supports the growth of MSC. While no significant differences were observed in the MSC numbers in swine cells cultured in the presence of FBS or CBS, human cells showed a greater proliferation potential in the presence of CBS as compared to FBS. Therefore, CBS can be used as an effective substitute to FBS for developing clinically useful protocols for culturing MSC.     

Inhibition of MEK/ERK signalling pathway promotes erythroid differentiation and reduces HSCs engraftment in ex vivo expanded haematopoietic stem cells

The MEK/ERK pathway is found to be important in regulating different biological processes such as proliferation, differentiation and survival in a wide variety of cells. However, its role in self‐renewal of haematopoietic stem cells is controversial and remains to be clarified. The aim of this study was to understand the role of MEK/ERK pathway in ex vivo expansion of mononuclear cells (MNCs) and purified CD34⁺ cells, both derived from human umbilical cord blood (hUCB). Based on our results, culturing the cells in the presence of an inhibitor of MEK/ERK pathway—PD0325901 (PD)—significantly reduces the expansion of CD34⁺ and CD34⁺ CD38^? cells, while there is no change in the expression of stemness‐related genes (HOXB4, BMI1). Moreover, in vivo analysis demonstrates that PD reduces engraftment capacity of ex vivo expanded CD34⁺ cells. Notably, when ERK pathway is blocked in UCB‐MNCs, spontaneous erythroid differentiation is promoted, found in concomitant with increasing number of burst‐forming unit‐erythroid colony (BFU‐E) as well as enhancement of erythroid glycophorin‐A marker. These results are in total conformity with up‐regulation of some erythroid enhancer genes (TAL1, GATA2, LMO2) and down‐regulation of some erythroid repressor genes (JUN, PU1) as well. Taken together, our results support the idea that MEK/ERK pathway has a critical role in achieving the correct balance between self‐renewal and differentiation of UCB cells. Also, we suggest that inhibition of ERK signalling could likely be a new key for erythroid induction of UCB‐haematopoietic progenitor cells.     

Chromosomal stability during ex vivo expansion of UCB CD34(+) cells

Objectives: Ex vivo expansion is a feasible strategy, which may overcome limitation of the very low frequency of haematopoietic stem/progenitor cells, in umbilical cord blood (UCB). However, both quality of cells and safety of expanded population are critical issues to be addressed for their clinical application. Hence, in this study, we evaluated genetic stability of UCB‐derived CD34⁺ cells during ex vivo culture, based on karyotype analysis, as well as its effect on cell proliferation characteristics. Materials and methods: CD34⁺ cells were isolated from human UCB samples by immunomagnetic separation and were expanded ex vivo over a 28‐day period. Expansion of total nucleate cells, CD34⁺ cells and CD34⁺ CD38^? cells was investigated. Karyotype analysis of the expanded cells from six randomly selected UCB samples was performed to evaluate their genetic stability. Results: Chromosomal abnormality of expanded cells mainly appeared by day 14, but was seldom sustained until day 28. None of the chromosomal abnormal samples displayed neoplastic proliferation, and expanded cells with altered chromosomes did not show obvious transformation phenomena according to soft agar assay. Conclusions: Ex vivo expansion could lead to occurrence of chromosomal abnormality, although here it did not produce excessive proliferative advantage of the expended cells. Importantly, chromosomal alteration seemed not to be inheritable and unlikely to result in malignant transformation. However, further in‐depth evaluation of potential clinical risks of chromosomal abnormality is warranted.     

Unrestricted somatic stem cells: interaction with CD34+ cells in vitro and in vivo, expression of homing genes and exclusion of tumorigenic potential

Background aimsTransplantation of allogeneic hematopoietic stem cells (HSC) within the framework of hematologic oncology or inherited diseases may be associated with complications such as engraftment failure and long-term pancytopenia. HSC engraftment can be improved, for example by co-transplantation with mesenchymal stem cells (MSC). Recently, a new multipotent MSC line from umbilical cord blood, unrestricted somatic stem cells (USSC), has been described. It was demonstrated that USSC significantly support proliferation of HSC in an in vitro feeder layer assay.MethodsA NOD/SCID mouse model was used to assess the effect of USSC on co-transplanted CD34⁺ cells and look for the fate of transplanted USSC. The migration potential of USSC was studied in a Boyden chamber migration assay and in vivo. Quantitative real-time polymerase chain reaction (qRT-PCR) for CXCR4, CD44, LFA1, CD62L, VLA4, RAC2, VLA5A and RAC1 were performed. NMR1 nu/nu mice were used for a tumorigenicity test.ResultsAfter 4 weeks, homing of human cells (CD45⁺) to the bone marrow of NOD/SCID mice was significantly increased in mice co-transplanted with CD34⁺ cells and USSC (median 30.9%, range 7–50%) compared with the CD34⁺ cell-only control group (median 5.9%, range 3–10%; P = 0.004). Homing of USSC could not be shown in the bone marrow. A cell–cell contact was not required for the graft enhancing effect of USSC. An in vivo tumorigenicity assay showed no tumorigenic potential of USSC.ConclusionsThis pre-clinical study clearly shows that USSC have an enhancing effect on engraftment of human CD34⁺ cells. USSC are a safe graft adjunct.     

Gatekeeper role of brain antigen‐presenting CD11c+ cells in neuroinflammation

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen‐presenting cells, dendritic cells. Applying intravital two‐photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen‐presenting CD11c⁺ cells, which preferentially interact with Th17 cells. IL‐17 expression correlates with expression of GM‐CSF by T cells and with accumulation of CNS CD11c⁺ cells. These CD11c⁺ cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c⁺ cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c⁺ cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS.     

Mesenchymal stromal cell exosome–enhanced regulatory T-cell production through an antigen-presenting cell–mediated pathway

Background aims

The immunomodulatory property of mesenchymal stromal cell (MSC) exosomes is well documented. On the basis of our previous report that MSC exosomes increased regulatory T-cell (Treg) production in mice with allogenic skin graft but not in ungrafted mice, we hypothesize that an activated immune system is key to exosome-mediated Treg production.

Directly auto‐transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto‐transplanted versus in vitro expanded MSC with or without bone morphogenetic protein‐2 (BMP‐2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto‐transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT‐PCR analysis before subcutaneous implantation in combination with BMP‐2 and β‐tricalcium phosphate/hydroxyapatite (β‐TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT‐PCR evaluation. Sheep MSC were CD29⁺, CD44⁺ and CD166⁺ after selection by Ficoll gradient centrifugation, while directly auto‐transplanted MSC‐populations expressed CD29 and CD166 at lower levels. Both, directly auto‐transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto‐transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β‐TCP/HA matrix comparable to the application of 60 μg/ml BMP‐2 only or implantation of expanded MSC. Bone matrix proteins were up‐regulated in constructs following direct auto‐transplantation and in expanded MSC as well as in BMP‐2 constructs. Up‐regulation was detected using immunohistology methods and RT‐PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto‐transplanted or expanded MSC with β‐TCP/HA granules alone. Hence BMP‐2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.