Morphological profiling using machine learning reveals emergent subpopulations of interferon-γ–stimulated mesenchymal stromal cells that predict immunosuppression

Background

Although a preponderance of pre-clinical data demonstrates the immunosuppressive potential of mesenchymal stromal cells (MSCs), significant heterogeneity and lack of critical quality attributes (CQAs) based on immunosuppressive capacity likely have contributed to inconsistent clinical outcomes. This heterogeneity exists not only between MSC lots derived from different donors, tissues and manufacturing conditions, but also within a given MSC lot in the form of functional subpopulations. We therefore explored the potential of functionally relevant morphological profiling (FRMP) to identify morphological subpopulations predictive of the immunosuppressive capacity of MSCs derived from multiple donors, manufacturers and passages.

Influence of hypoxia on the domiciliation of Mesenchymal Stem Cells after infusion into rats: possibilities of targeting pulmonary artery remodeling via cells therapies?

Background

Bone marrow (BM) cells are promising tools for vascular therapies. Here, we focused on the possibility of targeting the hypoxia-induced pulmonary artery hypertension remodeling with systemic delivery of BM-derived mesenchymal stem cells (MSCs) into non-irradiated rats.

Methods

Six-week-old Wistar rats were exposed to 3-week chronic hypoxia leading to pulmonary artery wall remodeling. Domiciliation of adhesive BM-derived CD45^- CD73⁺ CD90⁺ MSCs was first studied after a single intravenous infusion of Indium-111-labeled MSCs followed by whole body scintigraphies and autoradiographies of different harvested organs. In a second set of experiments, enhanced-GFP labeling allowed to observe distribution at later times using sequential infusions during the 3-week hypoxia exposure.

Results

A 30% pulmonary retention was observed by scintigraphies and no differences were observed in the global repartition between hypoxic and control groups. Intrapulmonary radioactivity repartition was homogenous in both groups, as shown by autoradiographies. BM-derived GFP-labeled MSCs were observed with a global repartition in liver, in spleen, in lung parenchyma and rarely in the adventitial layer of remodeled vessels. Furthermore this global repartition was not modified by hypoxia. Interestingly, these cells displayed in vivo bone marrow homing, proving a preservation of their viability and function. Bone marrow homing of GFP-labeled MSCs was increased in the hypoxic group.

Conclusion

Adhesive BM-derived CD45^- CD73⁺ CD90⁺ MSCs are not integrated in the pulmonary arteries remodeled media after repeated intravenous infusions in contrast to previously described in systemic vascular remodeling or with endothelial progenitor cells infusions.     

CD4+CD25+ regulatory T cells are not required for mesenchymal stem cell function in fully MHC-mismatched mouse cardiac transplantation

Although the immunomodulative properties of mesenchymal stem cells (MSCs) open up attractive possibilities in solid-organ transplantation, information concerning the optimal dose, route, timing of administration, major histocompatibility complex (MHC)-restriction and relevant mechanisms is currently lacking. Therefore, better characterization of MSC immunoregulatory activity and elucidation of its mechanisms are crucial. In this study, we confirmed that MSCs did not elicit proliferation by allogeneic CD4⁺ T cells, suggesting that MSCs were not immunogenic. By using C57BL/6 mouse MSCs as donor-derived or recipient-derived or as third-party MSCs, we discovered that MSCs suppressed CD4⁺ T cell proliferation and prolonged mouse cardiac allograft survival in a dose-dependent and non-MHC-restricted manner. We also found that intraperitoneal administration favored survival prolongation, although this prolongation was weaker than that via the intravenous route. Only infusion at earlier time points favored survival prolongation. Depletion of CD4⁺CD25⁺ T cells did not affect the immunosuppression of MSCs on CD4⁺ T cells. Moreover, MSCs did not induce regulatory T cells. The in vivo data revealed that MSCs did not increase the percentage of CD4⁺CD25⁺ T cells and FoxP3 expression. More importantly, we demonstrated for the first time that depletion of CD4⁺CD25⁺ T cells did not hinder MSC-induced survival prolongation, indicating that CD4⁺CD25⁺ regulatory T cells were not essential for the prolongation of MSC-mediated allograft survival.     

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSC) with similar properties to bone marrow-derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We have evaluated methods for culturing human limbal MSC (L-MSC).MethodsFour basic strategies were compared: serum-supplemented medium (10% fetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor and fibroblast growth factor 2, or one of two commercial serum-free media, defined keratinocyte serum-free medium (Invitrogen) and MesenCult-XF® (Stem Cell Technologies). The resulting cultures were examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141 and CD271), immunocytochemistry (alpha-smooth muscle actin; α-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis) and co-culture experiments with human limbal epithelial (HLE) cells.ResultsWhile all techniques supported the establishment of cultures to varying degrees, sustained growth and serial propagation were only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70–80% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, approximately 25% α-sma⁺ and displayed multipotency. Cultures established in MesenCult-XF were > 95% CD34^? CD45^? CD90⁺ CD73⁺ CD105⁺, 40% CD141⁺, rarely expressed α-sma, and displayed multipotency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ?Np63, along with the corneal differentiation marker cytokeratin 3.ConclusionsMesenCult-XF is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.     

Human endometrial stem cells: High-yield isolation and characterization

Background

Menstrual blood is only recently and still poorly studied, but it is an abundant and noninvasive source of highly proliferative mesenchymal stromal cells (MSCs). However, no appropriate isolation method has been reported due to its high viscosity and high content of clots and desquamated epithelium.

Endothelial origin of mesenchymal stem cells

Mesenchymal stem/stromal cells (MSCs) are fibroblastoid cells capable of long-term expansion and skeletogenic differentiation. While MSCs are known to originate from neural crest and mesoderm, immediate mesodermal precursors that give rise to MSCs have not been characterized. Recently, using human embryonic stem cells (hESCs), we demonstrated that mesodermal MSCs arise from APLNR⁺ precursors with angiogenic potential, mesenchymoangioblasts, which can be identified by FGF2-dependent colony-forming assay in serum-free semisolid medium. In this overview we provide additional insights on cellular pathways leading to MSC establishment from mesoderm, with special emphasis on endothelial-mesenchymal transition as a critical step in MSC formation. In addition, we highlight an essential role of FGF2 in induction of angiogenic cells with potential to transform into MSCs (mesenchymoangioblasts) or hematopoietic cells (hemangioblasts) from mesoderm, and discuss correlations of our in vitro findings with the course of angioblast development during embryogenesis.Key words: mesenchymoangioblast, hemangioblast, human embryonic stem cells, endothelial-mesenchymal transition, epithelial-mesenchymal transition, mesenchymal stem cells, endothelial cells, apelin receptor, FGFMesenchymal stem/stromal cells (MSCs) are defined as multipotent fibroblastoid cells that give rise to cells of the skeletal connective tissue including osteoblasts, chondrocytes and adipocytes.^1–4 Although MSCs were described more than 40 years ago and are widely used for cellular therapies, very little knowledge exists regarding the developmental origins of MSCs in the embryo, the hierarchy of MSC progenitors or heterogeneity of MSCs within tissues. It has been demonstrated that during embryonic development, MSCs arise from a two major sources: neural crest and mesoderm.^5–7 Using Cre-recombinase lineage tracing experiments, Takashima et al. identified Sox1⁺ neuroepithelium as pre-cursors of MSCs of neural crest origin. However, direct precursors of mesoderm-derived MSCs were unknown. To identify these precursors, we employed human embryonic stem cells (hESCs) directed toward mesendodermal differentiation in coculture with mouse bone marrow stromal cells OP9,⁸ using the experimental approach depicted in Figure 1. As shown in this differentiation system, mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by expression of apelin receptor (APLNR), otherwise known as angiotensin receptor like-1 receptor. Because we observed a positive selective effect of FGF2 on production of mesenchymal cells from hESCs in OP9 coculture, we decided to test whether FGF2 can induce the formation of colonies with mesenchymal potential from APLNR⁺ mesodermal cells. Indeed, when we isolated APLNR⁺ cells from hESCs differentiated on OP9 for 2 days and placed them in serum-free semisolid medium containing FGF2, we observed the formation of sharply-circumscribed spheroid colonies formed by tightly packed cells with a gene expression profile representative of embryonic mesenchyme originating from lateral plate/extraembryonic mesoderm and CD140a⁺CD146⁺C D90⁺CD56⁺CD166⁺CD31⁻CD43⁻CD45⁻ phenotype typical of mesenchymal cells. Based on cellular composition, we designated these colonies as mesenchymal (MS) colonies and cells forming these colonies as MS colony-forming cells (MS-CFCs). MS colony formation required serum-free medium and was solely dependent on FGF2 as a colony-forming factor. MS colonies were significantly enhanced by PDGF-BB, but suppressed by VEGF, TGFβ1 and Activin A. When transferred to the adherent cultures in serum-free medium with FGF2, individual MS colonies gave rise to multi-potential mesenchymal cell lines with typical phenotype (CD146⁺ CD105⁺ CD73⁺ CD31⁻ CD43/45⁻), differentiation (chondro-, osteo- and adipogenesis) and robust proliferation (>80 doublings) potentials. Using single cell deposition assay, chimeric hESC lines and time-lapse studies we demonstrated the clonality/single cell origin of MS colonies.Open in a separate windowFigure 1Schematic diagram of the experimental approach used to identify precursors and cellular events leading to formation of mesoderm-derived MSCs. hESCs were committed to mesendodermal differentiation through coculture with OP9 for 2 days. APLNR⁺ mesodermal cells were selected using magnetic sorting. In serum-free semisolid medium, APLNR⁺ cells grew into FGF2-dependent compact spheroid colonies composed of mesenchymal cells. MS colonies were formed through establishment of tightly-packed single cell-derived cores (day 3 of clonogenic culture), which expanded into spheroid colonies (days 6 and 12 of clonogenic culture). To evaluate differentiation potential, MS colonies were collected at different stages of clonogenic culture and placed on OP9. The presence of endothelial and mesenchymal cells after coculture of MS colonies with OP9 was evaluated by flow cytometry and immunofluorescence. In addition, colonies at core stage (day 3 of clonogenic culture) and mature colonies (day 12 of clonogenic cultures) were collected for molecular profiling studies. To generate clonal MSC lines, individual mature colonies were plated on the collagen/fibronectin-coated plastic and cultured in presence of FGF2.MS-CFCs could be detected only transiently, with a major peak on day 2 of hESC differentiation and disappeared after 4 days of differentiation. Notably, MS-CFC activity was developed prior to the expression of CD73 and CD105 MSC markers and upregulation of MSC-related genes, i.e., before onset of mesenchymogenesis. APLNR⁺ cells isolated from hESC cultures differentiated for 3 days also formed colonies in response to FGF2; however, the vast majority of these colonies were composed of blood cells and had a morphology similar to the previously described blast (BL) or hemangioblast colonies, which identify a common precursor for hematopoietic and endothelial cells.^9,10To fully evaluate the differentiation potential of MS colonies, we collected these colonies from semisolid cultures and placed them back on OP9 feeders, which are known to support development of a broad range of mesodermal lineage cells, including hematopoietic, vascular and cardiac.^11–13 Using this approach, we confirmed that individual BL colonies possess hemangioblastic potential, i.e., generate both hematopoietic and endothelial cells. When MS colonies were picked from clonogenic cultures and cultured on OP9, we found that the majority of cells differentiated into CD146⁺CD31⁻CD43/CD45⁻ mesenchymal cells as expected. However, we also discovered that MS colonies gave rise to CD31/VE-cadherin⁺CD43/45⁻ endothelial cells, indicating that MS colonies similar to BL colonies possess endothelial potential. The endothelial potential of MS colonies was also confirmed by demonstration of tube formation by MS colonies grown on Matrigel. In contrast, MSC lines derived from MS colonies did not produce any endothelial cells after coculture with OP9 indicating a progressive restriction of differentiation potential following MSC formation. Because single MS-CFC shows potential to form endothelium and MSCs, we designated the MSC precursor identified by this colony-forming assay as mesenchymoangioblast.To define more precisely the cellular events leading to establishing MSCs, we examined the formation of MS colonies using time-lapse cinematography and analyzed the kinetic of their angiogenic potential. As demonstrated by time-lapse studies, APLNR⁺ mesodermal cells placed in semisolid medium possessed a high motility, which was more pronounced before and during the first cell division. Following several divisions, single APLNR⁺ cells formed a core, an immotile structure composed of a small number of tightly packed cells. While APLNR⁺ mesodermal cells lacked endothelial gene expression, molecular profiling of MS colonies at the core stage revealed that these cells acquired angioblastic gene expression profile as indicated by upregulation of FLT1, TEK, CDH5 (VE-cadherin), PECAM1 (CD31), FLI1, SELE (ELAM-1) and ICAM2 endothelial genes. When we collected MS cores (day 3 of clonogenic culture) and placed them on OP9, they formed predominantly VE-cadherin⁺ endothelial clusters, strongly indicating the endothelial nature of the core-forming cells. Subsequently, cells at the periphery of the core underwent endothelial-mesenchymal transition (EndMT) and formed a shell of tightly packed spindle-like cells around the core. When we picked colonies at this stage (day 6 of colony-forming culture) and placed them on OP9, most of the colonies (>70%) grew cell clusters composed of endothelial and mesenchymal cells. In contrast, mature MS colonies collected on day 12 of clonogenic culture formed predominantly clusters of mesenchymal cells, indicating a progressive loss of endothelial potential following colony maturation. Although no CD31 expression was detected in the mesenchymal cells composing mature MS colonies, these cells retained several endothelial traits including surface expression of endothelial tyrosine kinase (TEK or TIE2), FLT1 (VEGFR1) and endomucin. The critical role of EndMT in MS colony formation and MSC development was also congruous with our observation of the suppressive effect of VEGF, a known inhibitor of EndMT,^14,15 on MS colonies. When VEGF was added to MS clonogenic cultures, hESC-derived mesodermal cells were capable of forming angiogenic cores; however, these cores did not transform into mesenchymal cells, indicating that VEGF abrogates MS colony development at the core stage through inhibition of EndoMT. The schematic diagram demonstrating development of mesodermal MSCs is presented in Figure 2.Open in a separate windowFigure 2A model of mesoderm-derived MSC development from hESCs. Coculture with OP9 stromal cells predominantly induces hESC differentiation toward APLNR⁺ mesoderm. APLNR⁺ population contains angiogenic mesodermal precursors with either mesenchymal (mesenchymoangioblast) or hematopoietic (hemangioblast) potentials. Mesenchymoangioblasts and hemangioblasts arise sequentially during differentiation and can be revealed by MS and BL colony formation in response to FGF2. Development of MS and BL colonies in semisolid media proceed through a core stage at which APLNR⁺ cells form clusters of tightly packed cells with angiogenic potential. Subsequently, core-forming cells undergo EndMT giving rise to mesenchymal cells, which form a shell around the core developing into a mature MS colony. VEGF, EndMT inhibitor, blocks MS colony-formation at core stage. The ability of MS-CFCs to generate mesenchymal and endothelial cells can be revealed by coculture of individual colonies with OP9. Similar to MS colonies, BL colonies are formed through establishment of angiogenic core. However, hemangioblast core-forming cells undergo endothelial-hematopoietic transition and grew hematopoietic cells around the core.The close relationship between endothelial and hematopoietic cell development was recognized more than 130 years ago (reviewed by ref. ¹⁶) and confirmed in multiple modern studies.^9,17–22 However, the association between endothelial pre-cursors and MSCs during development was not well established, although cells with endothelial and mural cell potential were identified²³ and the critical role of EndMT in the formation of endocardial cushion²⁴ and testicular cords²⁵ in the embryo was acknowledged. Our hESC-based in vitro studies indicated that formation of mesodermal MSCs proceed through the endothelial stage and likely included at least two successive cycles of cell transitions. Initially APLNR⁺ mesoderm, which consists of fibroblast-like migratory cells, give rise to core structures composed of tightly packed endothelial cells in response to FGF2. Subsequently, endothelial cells forming cores undergo epithelial-mesenchymal transition, i.e., EndMT and form MSCs. The question remains how well this in vitro model reflects in vivo development. Although only sparse data exist regarding MSC precursors in the embryo, development of angiogenic hematopoietic precursors, hemangioblasts was studied more extensively in mammals and birds, and therefore parallels between in vivo and in vitro studies can be drawn. As we demonstrated,⁸ APLNR⁺ mesodermal cells collected from hESCs differentiated on OP9 for 3 days formed disperse BL colonies that identify hemangioblasts in vivo and in vitro.^9,26 Similar to MS colonies, the development of BL colonies required FGF2 and proceeded through angiogenic core formation. However, in contrast to MS cores, BL cores transformed into blood cells, i.e., underwent endothelial-hematopoietic transformation (see Fig. 2). Importantly, in vivo studies identified FGF2 as the essential factor in hemangioblast induction²⁷ analogous to our in vitro observation. In chicken embryo, the activation of FGF signaling leads to aggregation of migrating mesodermal cells adjacent to the endoderm, upregulation of VEGFR2 (KDR) expression, and subsequent formation of angioblasts and hemangioblasts.^28–30 This sequence of events leading to hemangioblast development in vivo considerably resembles what we observed in vitro, and highly suggests accurate recapitulation of embryonic development by our hESC differentiation model. Therefore, searching for an in vivo equivalent of mesenchymonagioblast would be a reasonable next step.In addition to embryonic development, EndMT is also implicated in several pathologies including cancer progression and development of cardiac and renal fibrosis.^31–34 Recently, Olsen group revealed that endothelial cells could be transformed directly into MSCs through overexpression of ALK2 or its activation by TGFβ2 or BMP4,¹⁵ indicating that endothelial cells could be an important source of MSCs in postnatal life. Conversely, the transition from MSCs to endothelial cells, has been also described in reference ³⁵. Based on these observations, a cycle of cell-fate transition from endothelium to MSCs and back to endothelium was proposed as a circuit controlling stem cell state.³⁶ Since multiple parallels could be drawn between EndMT described in adult tissues and during hESC differentiation, one may wonder whether bipotential cells with endothelial and MSC potential similar to embryonic mesenchymoangioblasts are present and constitute an important element of EndMT circuit in adults.In conclusion, the identification of mesenchymoangioblast as a clonogenic precursor of mesoderm-derived MSCs is an important step toward defining pathways of MSC development and specification. In addition, the demonstration of MSC formation from mesoderm through EndMT provides new insights into the mechanisms involved in establishment of MSCs.     

Substance P enhances mesenchymal stem cells-mediated immune modulation

Since clinical application of MSCs requires long-term ex vivo culture inducing senescence in MSCs and reducing the therapeutic activity of transplanted MSCs, numerous efforts have been attempted to sustain the active state of MSCs. Substance P (SP) is a neuropeptide that functions to activate the cellular physiological responses of MSCs, including proliferation, migration, and secretion of specific cytokines. In this study, we explored the potential of SP to restore the weakened immune modulating activity of MSCs resulting from long-term culture by measuring T cell activity and interleukin-2 (IL-2) secretion of CD4⁺ Jurkat leukemic T cells and primary CD4⁺ T cells. As the number of cell passages increased, the immunosuppressive function of MSCs based on T cell activity decreased. This weakened activity of MSCs could be restored by SP treatment and nullified by co-treatment of an NK1 receptor blocker. Higher levels of transforming growth factor beta 1 (TGF-β1) secretion were noted in the medium of SP-treated late passage MSC cultures, but IL-10 levels did not change. SP-treated MSC-conditioned medium decreased T cell activity and IL-2/Interferon gamma (IFN-g) secretion in T cells even in the activation by lipopolysaccharide (LPS) or CD3/CD28 antibodies, both of which were successfully blocked by inhibiting the TGF beta signaling pathway. This stimulatory effect of SP on late passage MSCs was also confirmed in direct cell–cell contact co-culture of MSCs and CD4⁺ Jurkat T cells. Collectively, our study suggests that SP pretreatment to MSCs may recover the immunosuppressive function of late passage MSCs by potentiating their ability to secrete TGF-β1, which can enhance the therapeutic activity of ex vivo expanded MSCs in long-term culture.     

Back Cover Image,Volume 117, Number 1, January 2020

In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM-MSC) cultures. Sorted CD264⁺ hBM-MSCs from two age-matched donors have elevated β-galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264⁻ hBM-MSCs. Counterintuitive to their aging phenotype, CD264⁺ hBM-MSCs exhibited comparable survival to matched CD264⁻ hBM-MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony-forming efficiency. These findings have ramifications for the preparation of hBM-MSC therapies given the prevalence of aging CD264⁺ cells in hBM-MSC cultures and the popularity of colony-forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.     

Mesodermal progenitor cells (MPCs) differentiate into mesenchymal stromal cells (MSCs) by activation of Wnt5/calmodulin signalling pathway

Background

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4⁺CD105⁺CD90^neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4^negCD105⁺CD90^bright and MSCA-1⁺), in the primary cultures, resulted lower than 2%.

Methodology/Principal Finding

We demonstrate that MPCs differentiate to MSCs through an SSEA-4⁺CD105⁺CD90^bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4^neg). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ = 0.5 µM, p<0.01), while endothelial differentiation was unaffected.

Conclusion

The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.     

CD11c+ Cells Partially Mediate the Renoprotective Effect Induced by Bone Marrow-Derived Mesenchymal Stem Cells

Previous studies have shown that induction of immune tolerance by mesenchymal stem cells (MSCs) is partially mediated via monocytes or dendritic cells (DCs). The purpose of this study was to determine the role of CD11c⁺ cells in MSC-induced effects on ischemia/reperfusion injury (IRI). IRI was induced in wildtype (WT) mice and CD11c⁺-depleted mice following pretreatment with or without MSCs. In the in-vitro experiments, the MSC-treated CD11c⁺ cells acquired regulatory phenotype with increased intracellular IL-10 production. Although splenocytes cocultured with MSCs showed reduced T cell proliferation and expansion of CD4⁺FoxP3⁺ regulatory T cells (Tregs), depletion of CD11c⁺ cells was associated with partial loss of MSCs effect on T cells. In in-vivo experiment, MSCs’ renoprotective effect was also associated with induction of more immature CD11c⁺ cells and increased FoxP3 expression in I/R kidneys. However all these effects induced by the MSCs were partially abrogated when CD11c⁺ cells were depleted in the CD11c⁺-DTR transgenic mice. In addition, the observation that adoptive transfer of WT CD11c⁺ cells partially restored the beneficial effect of the MSCs, while transferring IL-10 deficient CD11c⁺ cells did not, strongly suggest the important contribution of IL-10 producing CD11c⁺ cells in attenuating kidney injury by MSCs. Our results suggest that the CD11c⁺ cell-Tregs play critical role in mediating renoprotective effect of MSCs.     

Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23-25 years

Objective

To evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs).

Methods

Samples of human BMCs that were cryopreserved for 23–25 years (n = 20) were thawed to obtain an initial culture and a primary culture (P₀) that was propagated through five passages (P₁–P₅) to obtain MSCs. Freshly collected human bone marrow samples (n = 20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P₃ cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing.

Results

In the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p < 0.05) while the relative rate of recovery of MSCs was only 48.5 ± 8.6% in P₀. At the end of P₃, fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p > 0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P₃ from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells.

Conclusion

Using the methods described here, long-term (23–25 years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.     

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.     

Isolation of adipose tissue mesenchymal stem cells without tissue destruction: A non-enzymatic method

The conventional enzymatic method is widely used for mesenchymal stem cells (MSCs) isolation from adipose tissue. The method holds major drawbacks; it is costly, time-consuming and results in a heterogeneous cell population. Besides, digestion of extracellular matrix causes cell injury and compromise proliferation and differentiation of the cells. Also, because of over handling the samples are also prone to contamination. Here, we introduce a non-enzymatic method for MSCs isolation without disturbing the cells habitat. Small pieces of adipose tissue obtained from animal or human liposuction were explanted into a culture flask, immobilized by fetal bovine serum (FBS) and incubated overnight. The explants were then irrigated with DMEM containing FBS. Within few days, the fibroblast-like cells migrated from the tissue and proliferated rapidly. When subconfluent, the cells were harvested, expanded through 3 passages and used for immunophenotyping and differentiation assays. As judged by flow cytometric analysis of surface markers (CD44⁺, CD105⁺, CD34⁻, CD45⁻), Oil Red O and Alizarin Red staining, the MSCs isolated by our non-enzymatic method were pluripotent and exhibited the potential for differentiation into adipocyte and osteoblast. Great isolation yields, homogeneity of isolated cells, brief procedure, and high economy are the advantages of our method over the conventional protocol.     

Hematopoietic stem cell and mesenchymal stem cell population size in bone marrow samples depends on patient’s age and harvesting technique

Mesenchymal stem cells (MSCs) are heterogeneous population of cells with great potential for regenerative medicine. MSCs are relatively easy to expand in a cell culture, however determination of their concentration in harvested tissue is more complex and is not implemented as routine procedure. To identify MSCs collected from bone marrow we have used two combinations of cell markers (CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺) and fibroblast colony-forming unit (CFU-F) assay. Further, in donors of various ages, mesenchymal stem cell concentration was compared with the result of CFU-F assay and with hematopoietic stem cell concentration, determined by a standardized flow cytometric assay. A positive correlation of MSC populations to the CFU-F numbers is observed, the population of the CD45^?/CD271⁺ cells correlates better with CFU-F numbers than the population of the CD45^?/CD73⁺/CD90⁺/CD105⁺ cells. The relationship between the hematopoietic CD45^dim/CD34⁺ cell concentration and mesenchymal CFU-Fs or CD45^?/CD271⁺ cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45^dim/CD34⁺, mesenchymal CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺ stem cells, and CFU-F numbers were noted. Additionally, statistically significant higher CFU-F numbers were observed when bone marrow samples were harvested from three different sites from the anterior iliac crest instead of harvesting the same sample amount only from one site.     

Isolation murine mesenchymal stem cells by positive selection

Isolation and purification of mesenchymal stem cells (MSCs) from mouse via plastic adherent cultures is arduous because of the unwanted growth of hematopoietic cells and non-MSCs. In this work, homogenous populations of CD34⁺ MSCs from mouse bone marrow were isolated via positive selection. For this purpose, C57Bl/6 mice were killed and bone marrow cells were aspirated before incubation with magnetic bead conjugated to anti-CD34 antibody. A sample of positively selected CD34⁺ cells were prepared for flow cytometry to examine the expression of CD34 antigen and others were subcultured in a 25-cm² culture flask. To investigate the mesenchymal nature, the plastic adherent cultivated cells were induced to differentiate along osteoblastic and adipogenic lineages. Furthermore, the expression of some surface markers was investigated by flow cytometry. According to the result, purified populations of fibroblast-like CD34⁺ cells were achieved in the first passage (1 wk after culture initiation). The cells expressed CD34, CD44, Sca-1, and Vcam-1 antigens (markers) but not CD11b and CD45. They were capable of differentiating into osteocytes and adipocytes. This study indicated that our protocol can result in the efficient isolation of homogenous populations of MSCs from C57BL/6 mouse bone marrow. We have shown that murine bone marrow-derived CD34⁺ cells with plastic adherent properties and capability of differentiating into skeletal lineages in vitro are MSCs.     

Humoral Activity of Cord Blood-Derived Stem/Progenitor Cells: Implications for Stem Cell-Based Adjuvant Therapy of Neurodegenerative Disorders

Background

Stem/progenitor cells (SPCs) demonstrate neuro-regenerative potential that is dependent upon their humoral activity by producing various trophic factors regulating cell migration, growth, and differentiation. Herein, we compared the expression of neurotrophins (NTs) and their receptors in specific umbilical cord blood (UCB) SPC populations, including lineage-negative, CD34⁺, and CD133⁺ cells, with that in unsorted, nucleated cells (NCs).

Methods and Results

The expression of NTs and their receptors was detected by QRT-PCR, western blotting, and immunofluorescent staining in UCB-derived SPC populations (i.e., NCs vs. lineage-negative, CD34⁺, and CD133⁺ cells). To better characterize, global gene expression profiles of SPCs were determined using genome-wide RNA microarray technology. Furthermore, the intracellular production of crucial neuro-regenerative NTs (i.e., BDNF and NT-3) was assessed in NCs and lineage-negative cells after incubation for 24, 48, and 72 h in both serum and serum-free conditions. We discovered significantly higher expression of NTs and NT receptors at both the mRNA and protein level in lineage-negative, CD34⁺, and CD133⁺ cells than in NCs. Global gene expression analysis revealed considerably higher expression of genes associated with the production and secretion of proteins, migration, proliferation, and differentiation in lineage-negative cells than in CD34⁺ or CD133⁺ cell populations. Notably, after short-term incubation under serum-free conditions, lineage-negative cells and NCs produced significantly higher amounts of BDNF and NT-3 than under steady-state conditions. Finally, conditioned medium (CM) from lineage-negative SPCs exerted a beneficial impact on neural cell survival and proliferation.

Conclusions

Collectively, our findings demonstrate that UCB-derived SPCs highly express NTs and their relevant receptors under steady-state conditions, NT expression is greater under stress-related conditions and that CM from SPCs favorable influence neural cell proliferation and survival. Understanding the mechanisms governing the characterization and humoral activity of subsets of SPCs may yield new therapeutic strategies that might be more effective in treating neurodegenerative disorders.