Mesenchymal Stromal Cells Markers Research Articles

International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials

Mesenchymal stromal cells (MSCs) as a pharmaceutical for ailments characterized by pathogenic autoimmune, alloimmune and inflammatory processes now cover the spectrum of early- to late-phase clinical trials in both industry and academic sponsored studies. There is a broad consensus that despite different tissue sourcing and varied culture expansion protocols, human MSC-like cell products likely share fundamental mechanisms of action mediating their anti-inflammatory and tissue repair functionalities. Identification of functional markers of potency and reduction to practice of standardized, easily deployable methods of measurements of such would benefit the field. This would satisfy both mechanistic research as well as development of release potency assays to meet Regulatory Authority requirements for conduct of advanced clinical studies and their eventual registration. In response to this unmet need, the International Society for Cellular Therapy (ISCT) addressed the issue at an international workshop in May 2015 as part of the 21st ISCT annual meeting in Las Vegas. The scope of the workshop was focused on discussing potency assays germane to immunomodulation by MSC-like products in clinical indications targeting immune disorders. We here provide consensus perspective arising from this forum. We propose that focused analysis of selected MSC markers robustly deployed by in vitro licensing and metricized with a matrix of assays should be responsive to requirements from Regulatory Authorities. Workshop participants identified three preferred analytic methods that could inform a matrix assay approach: quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers and protein-based assay of secretome. We also advocate that potency assays acceptable to the Regulatory Authorities be rendered publicly accessible in an “open-access” manner, such as through publication or database collection.

224 CHARACTERIZATION OF EQUINE ENDOMETRIAL-DERIVED MESENCHYMAL STROMAL CELLS

Equine mesenchymal stromal cells (MSC) are mainly harvested from bone marrow and adipose tissue, requiring surgical procedures. Although human endometrium is known to harbor mesenchymal precursor cells, the presence of MSC in equine endometrium, a dynamic tissue, has not been investigated. This study reports for the first time the culture and characterisation of MSC from equine endometrium compared with equine bone marrow (BM)-derived MSC. Samples of equine endometrium (n = 6) and BM (n = 3) were collected postmortem. Endometrial tissue was digested using a dissociation medium containing collagenase I and DNase type I, and CD227 (mucin-1)-bound magnetic beads were utilised to separate epithelial (CD227+) from stromal (CD227–) cell fractions. Red blood cells from BM samples were excluded using a density gradient. All cell fractions were cultured in DMEM/F-12 containing 10% fetal bovine serum. After expansion, colony-forming unit (CFU) assay at passage 2, trilineage differentiation (adipogenic, chondrogenic, osteogenic), and flow cytometry analysis at passage 3/4 were performed for CD227– fractions and BM-MSC. Descriptive statistical analysis and 2-tailed t-test was performed with IBM SPSS Statistics 22 (SPSS Inc./IBM, Chicago, IL, USA). Both isolated cell fractions were plastic adherent and grew well under standard MSC culture conditions, although endometrial CD227– cells attached quicker to culture plasticware than did BM-MSC. The CFU assay at passage 2 showed no significant difference in cloning efficiency (CE) between BM-MSC (20.78 ± 2.86%) and CD227– (24.89 ± 3.04%) cell lines (P = 0.36). Flow cytometry showed the expression of MSC markers (CD29, CD44, CD90, CD105) and perivascular markers (CD146, NG2) but almost no expression of haematopoietic markers (CD34, CD45) in both cell lines (Table 1). No statistically relevant difference was seen except for the higher expression of NG2 in BM-MSC (P = 0.054). Trilineage differentiation was successfully induced in both cell lines. In conclusion, we showed the presence of putative MSC in equine endometrium. We successfully isolated and cultured these cells, which display comparable characteristics in MSC criteria as well-established BM-derived MSC. These endometrial-derived MSC may provide a convenient source for veterinary regenerative therapies in equine reproduction. Table 1.Flow cytometry marker expression (mean ± standard error) at passage 4

Phenotypic Change and Induction of Cytokeratin Expression During In Vitro Culture of Corneal Stromal Cells.

Cells of the corneal epithelium and stroma can be distinguished in vivo by different intermediate filaments, cytokeratins for corneal epithelial cells (CEC) and vimentin for keratocytes. Isolated and cultured keratocytes change phenotype, losing expression of keratocyte markers and gaining markers associated with mesenchymal stromal cells (MSC). This study investigates this change in phenotype in relation to intermediate filament expression in cultured corneal stromal cells (CSC) compared to CEC. Expression of epithelial markers (CK3, CK12, CK19, pan cytokeratin, E-cadherin), keratocyte markers (CD34, vimentin), and MSC markers (CD73, CD90, and CD105) were compared in CEC and CSC by immunocytochemistry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Expression was evaluated at different stages of CSC culture and compared to another stromal cell type, extracted from Wharton's jelly (WJ-MSC). In vivo keratocytes did not express cytokeratins. However, cultured CSC expressed epithelial-associated CK3, CK12, and CK19, but not other cytokeratins. Expression of cytokeratins increased as CSC were passaged and decreased as CSC were induced to become quiescent. Comparatively, WJ-MSC expressed lower levels of CK3, CK12, and CK19, but also stained for pan cytokeratin and expressed KRT5. Cultured CSC undergo phenotypic change during culture, expressing specific cytokeratin filaments normally associated with CEC. Cytokeratin expression begins as cells are cultured on plastic and increases with passage. This discovery may influence the way in which differences are discerned between cultured CEC and CSC. Investigators need to be aware that the expression of cytokeratins does not necessarily represent epithelial contamination, and that CEC and CSC may be more related than previously recognized.

Differentiation of Stem Cells From Human Exfoliated Deciduous Teeth Toward a Phenotype of Corneal Epithelium In Vitro.

The aim of this study was to characterize stem cells from human exfoliated deciduous teeth (SHED) and to investigate the potential of SHED to differentiate toward corneal epithelium-like cells in vitro. Mesenchymal and embryonic stem cell markers were analyzed by flow cytometry. The SHED was cocultured in either a transwell noncontact system or in a mixed culture system with immortalized human corneal epithelial (HCE-T) cells to induce the epithelial transdifferentiation. Expression of the mature corneal epithelium-specific marker cytokeratin 3 (CK3) and corneal epithelial progenitor marker cytokeratin 19 (CK19) were detected by immunofluorescence and the reverse transcription-polymerase chain reaction, respectively. SHED strongly expressed a set of mesenchymal stromal cell markers and pluripotency markers including NANOG and OCT-4. Seven days after the transwells were cocultured with HCE-T cells, SHED successfully upregulated epithelial lineage markers CK3 (16.6 ± 7.9%) and CK19 (10.0 ± 4.3%) demonstrating the potential for epithelial transdifferentiation, whereas CK3 and CK19 were barely expressed in SHED when cultured alone. Expression of transcript levels of CK3 and CK19 were significantly upregulated when SHED were transwell cocultured or mixed cultured with HCE-T cells by 7, 14, and 21 days. We have demonstrated that SHED retain the potential for transdifferentiation to corneal epithelium-like cells by in vitro coculture with immortal corneal epithelium cells. Thus, exfoliated teeth may be an alternative tissue resource for providing stem cells for potential clinical applications in ocular surface regeneration.

A new standardized clinical-grade protocol for banking human umbilical cord tissue cells.

Mesenchymal stromal cells (MSCs) isolated from human umbilical cord tissue (UCT) can be considered the perfect candidates for cell-based therapies and regenerative medicine. UCT-derived MSCs can be cryogenically stored in cell banks and expanded as needed for therapeutic uses. We developed a new method for UCT-MSC isolation, cryopreservation, and expansion, following all criteria required by a stem cell bank. UCT-MSCs were isolated either by manual dissociation (MM) or by a semiautomatic dissociation system (SAM). In both protocols UCTs were treated enzymatically using Type IV collagenase good manufacturing practices (GMP) graded and hyaluronidase (medicinal product). Isolated UCT-MSCs were cryopreserved and analyzed after thawing for phenotype; for proliferation rate; and for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. We found that SAM reduced the time of tissue enzyme exposure and enabled us to obtain a homogeneous single-cell suspension deprived of tissue fragments. The isolated cells in both groups showed high expression of MSC markers CD105, CD73, and CD90 and similar differentiation capabilities, phenotype, and proliferation potential. Moreover, the final yield of MSCs was comparable between the two techniques. In this study, we have established a reliable and standardized protocol to isolate UCT-MSCs from UCT for cell banking purposes. Processing the whole umbilical tissue with GMP-graded enzymes using a semiautomatic dissociator allowed us to obtain a single-cell suspension product with a known number of isolated cells that can be cryopreserved right after isolation and thawed as needed for expansion and clinical use.

The expression of neurogenic markers after neuronal induction of chorion-derived mesenchymal stromal cells

Objectives:Chorion is a tissue of early embryologic period that is discarded after delivery. It might be the potential source of mesenchymal stromal cells (MSCs) that can be used for research and eventually for therapeutic studies. At present, the biological properties and the differentiation capacity of chorion-derived MSCs are still poorly characterised. The objective of this study is to characterise and explore the differentiating potential of chorion-derived MSCs towards the neuronal lineages.Methods:Chorionic membrane was digested with enzyme and cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum. The expression of MSC markers was examined using flow cytometry. The adipogenic, osteogenic and neurogenic differentiation were examined by culturing in appropriate induction media. The expression of neuronal markers was determined by immunofluorescence and quantitative real time-PCR.Results:Chorion-derived MSCs were easily expanded up to 20 passages. They were positive for MSC markers (CD73, CD90 and CD105), and negative for haematopoietic markers (CD34 and CD45). Chorion-derived MSCs could differentiate into several mesodermal-lineages including adipocytes and osteoblasts. Moreover, chorion-derived MSCs could differentiate into neuronal-like cells as characterised by cell morphology and the presence of neural markers including MAP-2, glial fibrillary acidic protein (GFAP) and beta-tubulin III.Discussion:Chorion-derived MSCs can be readily obtained and expanded in culture. These cells also have transdifferentiation capacity as evidenced by their neuronal differentiation potential. Therefore, chorion can be used as an alternative source of MSCs for stem cell therapy in nervous system disorders.

BMP4: a Possible Key Factor in Differentiation of Auditory Neuron-Like Cells from Bone-Derived Mesenchymal Stromal Cells.

Previous studies have shown that BMP4 may play an important part in the development of auditory neurons (ANs), which are degenerated in sensorineural hearing loss. However, whether BMP4 can promote sensory fate specification from mesenchymal stromal cells (MSCs) is unknown so far. MSCs isolated from Sprague-Dawley (SD) rats were confirmed by expression of MSC markers using flow cytometry and adipogenesis/osteogenesis using differentiation assays. MSCs treated with a complex of neurotrophic factors (BMP4 group and non-BMP4 group) were induced into auditory neuron-like cells, then the differences between the two groups were analyzed in morphological observation, cell growth curve, qRT-PCR, and immunofluorescence. Flow cytometric analysis showed that the isolated cells expressed typical MSC surface markers. After adipogenic and osteogenic induction, the cells were stained by oil red O and Alizarin Red. The neuronal induced cells were in the growth plateau and had special forms of neurons. In the presence of BMP4, the inner ear genes NF-M, Neurog1, GluR4, NeuroD, Calretinin, NeuN, Tau, and GATA3 were up-regulated in MSCs. MSCs have the capacity to differentiate into auditory neuron-like cells in vitro. As an effective inducer, BMP4 may play a key role in transdifferentiation.

Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

In this study we have generated canine mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, from canine induced pluripotent stem cells (ciPSCs) by small-molecule inhibition of the transforming growth factor beta (TGFβ)/activin signaling pathway. These ciPSC-derived MSCs (ciPSC-MSCs) express the MSC markers CD73, CD90, CD105, STRO1, cPDGFRβ and cKDR, in addition to the pluripotency factors OCT4, NANOG and REX1. ciPSC-MSCs lack immunostaining for H3K27me3, suggesting that they possess two active X chromosomes. ciPSC-MSCs are highly proliferative and undergo robust differentiation along the osteo-, chondro- and adipogenic pathways, but do not form teratoma-like tissues in vitro. Of further significance for the translational potential of ciPSC-MSCs, we show that these cells can be encapsulated and maintained within injectable hydrogel matrices that, when functionalized with bound pentosan polysulfate, dramatically enhance chondrogenesis and inhibit osteogenesis. The ability to efficiently derive large numbers of highly proliferative canine MSCs from ciPSCs that can be incorporated into injectable, functionalized hydrogels that enhance their differentiation along a desired lineage constitutes an important milestone towards developing an effective MSC-based therapy for osteoarthritis in dogs, but equally provides a model system for assessing the efficacy and safety of analogous approaches for treating human degenerative joint diseases.

Tumor-driven Molecular Changes in Human Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) exert either tumor-stimulatory or tumor-inhibitory effect. The outcome of the tumor-MSC interaction is dictated by the tumor-specific activating signals. We analyzed the alterations in MSC phenotype in response to stimulation by tumor-secreted paracrine factors. Paracrine factors from human melanoma A375 and glioblastoma 8MGBA cells were used for prolonged culture of MSC to produce derived cells designated DIFF(A)-MSC or DIFF(G)-MSC, respectively. Derived cells were analyzed for the specific surface markers, the expression pattern of MSC markers and fibroblast-specific proteins. Changes in the cell phenotype were evaluated using scratch wound assay and tube formation in vitro; and xenotransplant growth in vivo. Our data show induced expression of vascular endothelial growth factor 2, CD146, fibroblast-specific protein, vimentin and endosialin in DIFF(A)-MSC cells. This indicates their differentiation towards the cells with features of tumor-associated fibroblasts upon stimulation with melanoma-secreted cytokines. Paracrine stimulation in DIFF(G)-MSC led to up-regulation of the genes involved in the MSC differentiation. MSC-specific surface marker characteristics were preserved in derived DIFF(A)-MSC and DIFF(G)-MSC cells. However, we observed increased proportion of CD146 and GD2 (neural ganglioside) positive cells and decreased expression of marker NG2 in the MSC exposed to tumor-conditioned medium. Melanoma-CM increased MSC migration, glioblastoma-CM compromised angiogenic capacity of MSC in vitro and the protumorigenic effect in vivo. Our data directly compare the pleiotropic effects mediated by the malignant cells on the MSC. Secreted paracrine factors from melanoma or glioblastoma differently changed molecular traits in MSC, which explains the dual role of MSC in tumor growth.

Osteoblastic differentiation of Wharton jelly biopsy specimens and their mesenchymal stromal cells after serum-free culture.

Cleft lip and cleft palate are increasingly being detected by prenatal ultrasound, which raises the opportunity of using the patient's own osteogenicity from umbilical cord mesenchymal cells for bony repair. The authors address the growth of the cells under a fully defined and regulated protocol. Wharton jelly-derived mesenchymal stromal cells were isolated and expanded as a monolayer with defined serum-free medium. Osteoblastic differentiation was tested in the cells and in the entire Wharton jelly biopsy specimens. The serum-free-cultured cells were included in hydroxyapatite granule-fibrin constructs and, without predifferentiation, subcutaneously implanted into immunoincompetent mice. Isolation and expansion of Wharton jelly-derived mesenchymal stromal cells were consistently successful under serum-free conditions, and the cells expressed standard mesenchymal stromal cell markers. The serum-free-cultivated cells produced a mineralized extracellular matrix under osteogenic differentiation, with a significant increase of osteoblastic lineage gene expression (Hox-A10 and Runx2) and an up-regulation of downstream osteogenic genes (OSX, OCN, ALPL, and BSP2). In vivo, they formed a dense matrix adjacent to the granules after 8 weeks, but no lamellar bone. serum-free-cultivated entire Wharton jelly biopsy specimens produced a mineralized extracellular matrix within the collagen matrix of the Wharton jelly. The osteogenic differentiation potential of Wharton jelly-derived mesenchymal stromal cells was maintained under serum-free isolation and expansion techniques. The cells without predifferentiation form a dense collagen matrix but not bone in vivo. Moreover, entire Wharton jelly biopsy specimens showed periosteal-like mineralization under osteogenic differentiation, which offers new options for autologous bone tissue engineering, including cleft palate surgery.

TEMPORARY REMOVAL: CD271 identifies functional human hepatic stellate cells, which localize in peri-sinusoidal and portal areas in liver after partial hepatectomy

Hepatic stellate cells (HSCs) are liver-resident mesenchymal cells involved in essential processes in the liver. However, knowledge concerning these cells in human livers is limited because of the lack of a simple isolation method. We isolated fetal and adult human liver cells by immunomagnetic beads coated with antibodies to a mesenchymal stromal cell marker (CD271) to enrich a population of HSCs. The cells were characterized by cell cultivation, immunocytochemistry, flow cytometry, reverse-transcription polymerase chain reaction and immunohistochemistry. Cells were injected into nude mice after partial hepatectomy to study in vivo localization of the cells. In vitro, CD271(+) cells were lipid-containing cells expressing several HSC markers: the glial fibrillary acidic protein, desmin, vimentin and α-smooth muscle actin but negative for CK8, albumin and hepatocyte antigen. The cells produced several inflammatory cytokines such as interleukin (IL)-6, IL-1A, IL-1B and IL-8 and matrix metalloproteinases MMP-1 and MMP-3 and inhibitors TIMP-1 and TIMP-2. In vivo, fetal CD271(+) cells were found in the peri-sinusoidal space and around portal vessels, whereas adult CD271(+) cells were found mainly in the portal connective tissue and in the walls of the portal vessels, which co-localized withα-smooth muscle actin or desmin. CD271(-) cells did not show this pattern of distribution in the liver parenchyma. The described protocol establishes a method for isolation of mesenchymal cell precursors for hepatic stellate cells, portal fibroblasts and vascular smooth muscle cells. These cells provide a novel culture system to study human hepatic fibrogenesis, gene expression and transcription factors controlling HSC regulation.

Myeloid-Derived Suppressor Cells are Generated during Retroviral Transduction of Murine Bone Marrow

Previous work by our group showed that transferring bone marrow cells transduced with an autoantigen into nonmyeloablated mice with experimental autoimmune encephalomyelitis induced immune tolerance and improved symptoms of the disease. Because this effect occurred in the absence of molecular chimerism, we hypothesized that the cells responsible did not have repopulating ability and that they were not mediating central but peripheral tolerance mechanisms. In the present study, we analyzed the immunophenotype of the cells that are generated in the transduction cultures and we evaluated the immunosuppressive activity of the main cell subpopulations produced. We show that both granulocytic (CD11b(+) Gr-1(hi)) and monocytic (CD11b(+) Gr-1(lo)) myeloid-derived suppressor cells (G- and M-MDSCs, respectively) are generated during standard 4-day γ-retroviral transduction cultures (representing about 25% and 40% of the total cell output, respectively) and that the effectively transduced cells largely consist of these two cell types. A third cell population representing about 15% of the transduced cells did not express CD45 or hematopoietic lineage markers and expressed mesenchymal stromal cell markers. Transduced total bone marrow cells and sorted M-MDSCs expressed arginase and inducible nitric oxide synthase activities, produced reactive oxygen species, and inhibited antigen-induced T-cell proliferation in vitro. Transgene-expressing MDSCs could be exploited therapeutically to induce tolerance in autoimmune diseases and in gene therapy protocols.

Role Of Long Pentraxin 3 (PTX3) In Wound Closure Induced By Bone Marrow-Derived Mesenchymal Stromal Cells

Although several studies have shown the capacity of mesenchymal stromal cells (MSCs) to repair and regenerate different tissues, the mechanisms underlying these processes are not understood. Long Pentraxin 3 (PTX3) is a multifunctional protein produced by MSCs and other cell subsets upon activation with inflammatory cytokines. PTX3 is involved in innate immunity, inflammation and extracellular matrix deposition. In the present study we analyzed the potential role of PTX3 in wound repair process induced by MSCs.PTX3 knockout MSCs (PTX3-/-MSCs) were collected from bone marrow of PTX3-/- mice. After 3-5 culture passages the expression of surface markers was analyzed by flow cytometry and their osteogenic and adipogenic differentiation capacity was detected by alizarin red O and oil red S staining, respectively. The ability of PTX3-/-MSCs to abrogate T cell proliferation was evaluated by co-culturing MSCs and PBMCs previously activated with Phytohaemagglutinin. Finally, equal number of both PTX3-/-MSCs and wild type (WT) MSCs were implanted into excisional wounds created by a biopsy punch on the back of allogenic WT and PTX3-/- mice. Wound area was measured up to 14 day and calculated using an image analysis program. The wound specimens were collected at 2, 7 and 14 days and processed for histological analysis.We demonstrated that PTX3-/-MSCs, similarly to WT MSCs, displayed typical fibroblastoid morphology, they expressed common MSC markers and were able to differentiate into adipocytes and osteoblasts. In addition, they drastically decreased the mitogen-induced proliferation of lymphocyte. Importantly, in a mouse model of wound healing, PTX3-/- MSCs showed a highly significant defect in wound closure compared to WT MSCs at each time point. Histological evaluation of skin samples treated with PTX3-/- MSCs showed a reduction of the granulation tissue and a significant increase of neutrophils (GR-1+) in the wound bed. Moreover, wounds treated with PTX3-/- MSCs were characterized by an excessive accumulation of fibrin at the 2nd day after injury. Accordingly, PTX3-/- MSCs showed a defective ability to degrade the fibrin matrix in vitro. Finally, PTX3-/- MSCs failed to close the ulcers in PTX3-/- mice.In conclusion, we demonstrated that PTX3 deficiency does not alter the phenotype and the capacity of MSCs to differentiate into mesengetic lineages; however, the production of PTX3 represents an essential requirement for MSC ability of enhancing tissue repair. Disclosures:No relevant conflicts of interest to declare.

The influence of bone marrow- and synovium-derived mesenchymal stromal cells from osteoarthritis patients on regulatory T cells in co-culture

There is increasing evidence that inflammation in the synovium plays a major role in the progression of osteoarthritis (OA). However, the immunogenic properties of mesenchymal stromal cells (MSCs), which are considered to regulate immunity in various diseases, remain largely unknown in OA. The purpose of this study was to determine the influence of MSCs from OA patients on regulatory T cells (Tregs ) in an allogeneic co-culture model. Bone marrow (BM) and synovial membrane (SM) were harvested from hip joints of OA patients and co-cultured with lymphocytes enriched in CD4(+) CD25(+) CD127(-) regulatory T cells (Treg (+) LC) from healthy donors. Treg proportions and MSC markers were assessed by flow cytometry. Cytokine levels were assessed after 2 and 5 days of co-cultivation. Additionally, Treg (+) LC cultures were analysed in the presence of interleukin (IL)-6 and MSC-supernatant complemented medium. B-MSCs and S-MSCs were able to retain the Treg proportion compared to lymphocyte monocultures. T cell-MSC co-cultures showed a significant increase of IL-6 compared to MSC cultures. S-MSCs produced higher amounts of IL-6 compared to B-MSCs, both in single and T cell co-cultures. The effect of retaining the Treg percentage could be reproduced partially by IL-6 addition to the medium, but could only be observed fully when using MSC culture supernatants. Our data demonstrate that retaining the Treg phenotype in MSC-T cell co-cultures can be mediated by MSC derived from OA patients. IL-6 plays an important role in mediating these processes. To our knowledge, this study is the first describing the interaction of MSCs from OA patients and Tregs in an allogeneic co-culture model.

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.

Long-term phenotypic characterization of human bone marrow and adipose tissue derived mesenchymal stromal cells

We present methods to characterize mesenchymal stromal cells (MSC) over long time periods in vitro. The methods entail passaging cells multiple times and performing differentiation studies with the cells at each passage. Using an array of surface markers and flow cytometric quantification, the data can be correlated to traditional measures of differentiation such as PCR and staining. Using these methods to quantify the amount of differentiation, we concluded that many common MSC markers do not specifically define MSCs with true stem cell properties. Additionally, adipose-derived as opposed to bone marrow-derived MSCs show long-term CD34+ labeling. The methods described can be used to help identify stem cell markers and to characterize the state of stem cells in vitro. Compiling these data from multiple laboratories would be helpful to determine source, extraction and culture methods needed to obtain high yields of useful stem cells.

Mesenchymal stromal cells of human umbilical cord Wharton's jelly accelerate wound healing by paracrine mechanisms

Mesenchymal 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. HUCPVC 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. HUCPVC 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. Our 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.

Distinctive contact between CD34+ hematopoietic progenitors and CXCL12+ CD271+ mesenchymal stromal cells in benign and myelodysplastic bone marrow

Mesenchymal stromal cells (MSCs) support hematopoiesis and are cytogenetically and functionally abnormal in myelodysplastic syndrome (MDS), implying a possible pathophysiologic role in MDS and potential utility as a diagnostic or risk-stratifying tool. We have analyzed putative MSC markers and their relationship to CD34+ hematopoietic stem/progenitor cells (HSPCs) within intact human bone marrow in paraffin-embedded bone marrow core biopsies of benign, MDS and leukemic (AML) marrows using tissue microarrays to facilitate scanning, image analysis and quantitation. We found that CD271+, ALP+ MSCs formed an extensive branching perivascular, periosteal and parenchymal network. Nestin was brightly positive in capillary/arteriolar endothelium and occasional subendothelial cells, whereas CD146 was most brightly expressed in SMA+ vascular smooth muscle/pericytes. CD271+ MSCs were distinct by double immunofluorescence from CD163+ macrophages and were in close contact with but distinct from brightly nestin+ and from brightly CD146+ vascular elements. Double immunofluorescence revealed an intimate spatial relationship between CD34+ HSPCs and CD271+ MSCs; remarkably, 86% of CD34+ HSPCs were in direct contact with CD271+ MSCs across benign, MDS and AML marrows, predominantly in a perivascular distribution. Expression of the intercrine chemokine CXCL12 was strong in the vasculature in both benign and neoplastic marrow, but was also present in extravascular parenchymal cells, particularly in MDS specimens. We identified these parenchymal cells as MSCs by ALP/CXCL12 and CD271/CXCL12 double immunofluorescence. The area covered by CXCL12+ ALP+ MSCs was significantly greater in MDS compared with benign and AML marrow (P=0.021, Kruskal–Wallis test). The preservation of direct CD271+ MSC/CD34+ HSPC contact across benign and neoplastic marrow suggests a physiologically important role for the CD271+ MSC/CD34+ HSPC relationship and possible abnormal exposure of CD34+ HSPCs to increased MSC CXCL12 expression in MDS.

GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC

BackgroundMesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components.Methods and FindingsWe established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform.ConclusionsThe five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells

The concept that mesenchymal stromal cells (MSCs), a component of the hematopoietic microenvironment, can be a target for alloreactive effector cells in the context of graft-vs-host disease has not been investigated in detail. Mixed lymphocyte reaction (MLR) supernatant was used to mimic the inflammatory milieu induced by an allogeneic immune response in vitro. In addition to phenotype and proliferation, we monitored MSC differentiation, gene expression, and support of CD34(+) hematopoietic stem and progenitor cells after priming with MLR supernatant. Priming of MSCs with MLR supernatant led to an 11-fold decrease in cobblestone area-forming cells in the 4-week coculture (p < 0.05) and a threefold decrease of colony-forming unit macrophage in the colony-forming cell assay (p < 0.05). MSC proliferation over 8 days was increased 2.5-fold (p < 0.05). Osteogenic differentiation was enhanced, while adipogenesis was concurrently suppressed. In addition, the surface expression of HLA-DR and intercellular adhesion molecule-1 was increased 20-fold (p = 0.06) and 45-fold (p < 0.05), respectively. This was associated with increased adhesion of hematopoietic stem and progenitor cells to MLR-treated MSCs. In summary, our data shed light on the dysfunction of the stromal environment during graft-vs-host disease, possibly aggravating cytopenia and leading to an enhanced immunogenicity of MSCs.