Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues

Mesenchymal stem cells (MSCs), which evoke only minimal immune reactivity, may have anti-inflammatory and immunomodulatory effects. In this study, we conducted a comparative analysis of the immunomodulatory properties of MSCs derived from adult human tissues including bone marrow (BM), adipose tissues (AT), umbilical cord blood (CB), and cord Wharton’s jelly (WJ). Using a multiple cytokine detection assay, we showed that there were no significant differences in levels of secreted factors from non-stimulated MSCs. We compared the immunosuppressive effect of BM-MSCs, AT-MSCs, CB-MSCs, and WJ-MSCs on phytohemagglutinin-induced T-cell proliferation. AT-MSCs, CB-MSCs, and WJ-MSCs effectively suppressed mitogen-induced T-cell proliferation as effectively as did BM-MSCs. Levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α secreted from activated T-cells increased over time, but these levels were significantly reduced when cocultured with each type of MSCs. In addition, the expression of hepatocyte growth factor, IL-10, transforming growth factor-β₁, cyclooxygenase (COX)-1, and COX-2 were unchanged in MSCs treated with IFN-γ and/or TNF-α, while indoleamine 2,3-dioxygenase (IDO) expression increased. IFN-γ and/or TNF-α produced by activated T-cells were correlated with induction of IDO expression by MSCs, which, in turn, suppressed T-cell proliferation. These findings suggest that MSCs derived from AT, CB, or WJ could be substituted for BM-MSCs for treatment of allogeneic conflicts.     

CD106 Identifies a Subpopulation of Mesenchymal Stem Cells with Unique Immunomodulatory Properties

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106⁺cells and CD106⁻cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106⁺CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106⁺CV-MSCs expressed more cytokines than CD106⁻CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.     

Impaired immunomodulatory function of chronic myeloid leukemia cancer stem cells and the possible mechanism involved in it

Backgroud

Cancer stem cells (CSCs) are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Development of specific therapies targeted at CSCs holds hope for the improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. This is particularly true in chronic myeloid leukemia (CML).

Methods

In this study, we isolated fetal liver kinase-1-positive (Flk1⁺) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph⁺) patients with stem cells property. We examined their biological characteristics as well as immunological function and further detected the possible molecular mechanism involved in the leukemia genesis.

Results

We showed that CML patient-derived Flk1⁺CD31^?CD34^? MSCs had normal morphology, phenotype and karyotype but appeared impaired immunomodulatory function. The capacity of Flk1⁺CD31^?CD34^? MSCs from CML patients to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have dampening immunomodulatory functions, suggesting that the dysregulation of hematopoiesis and immune response might originate from MSCs rather than HSCs. These Ph⁺ putative CML hemangioblast upregulated TGF-β1 and resultantly activated matrix metalloproteinase-9 (MMP-9) to enhance s-KitL and s-ICAM-1 secretion, which activated c-kit⁺ HSCs from the quiescent state to proliferative state. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was involved in CML pathogenesis.

Conclusions

Flk1⁺CD31^?CD34^? MSCs that express BCR/ABL leukemia oncogene are CSCs of CML and they play a critical role in the progression of CML through PI3K/Akt/NF-κB/MMP-9/s-ICAM-1/s-KitL signaling pathway beyond HSCs.     

Effect of priming of multipotent mesenchymal stromal cells with interferon γ on their immunomodulating properties

Multipotent mesenchymal stromal cells (MSCs) are widely used for cell therapy, in particular for prophylaxis and treatment of graft-versus-host disease. Due to their immunomodulatory properties, MSCs affect the composition of lymphocyte subpopulations, which depends on the immunological state of the organism and can change in different diseases and during treatment. Administration of MSCs is not always effective. Treatment of MSCs with different cytokines (in particular IFN-γ) leads to enhancement of their immunomodulatory properties. The aim of this study was to investigate sub-populational alterations and activation markers in lymphocytes (activated and non-activated) after interaction with MSCs and MSCs pretreated with IFN-γ (γMSCs) in vitro. Lymphocytes were co-cultured with MSCs or γMSCs for 4 days. The proportion of CD4⁺ and CD8⁺ expressing CD25, CD38, CD69, HLA-DR, and PD-1 and distribution of memory and effector subsets were measured by flow cytometry after co-cultivation of lymphocytes with MSCs or γMSCs. The distribution of lymphocyte subpopulations changes during culturing. In non-activated lymphocytes cultured without MSCs, decrease in the proportion of naïve cells and increase in the number of effector cells was observed. That could be explained as activation of lymphocytes in the presence of serum in culturing medium. Co-culturing of lymphocytes with MSCs and γMSCs leads to retention of their non-activated state. Activation of lymphocytes with phytohemagglutinin increases the number of central memory cells and activates marker expression. Interaction with MSCs and γMSCs prevents activation of lymphocytes and keeps their naïve state. Priming with IFN-γ did not induce MSCs inhibitory effect on activation of lymphocytes.     

CCR5 Antagonism Impacts Vaccination Response and Immune Profile in HIV-1 Infection

Maraviroc (MVC) is the first licensed antiretroviral therapeutic agent to target a host cell surface molecule, and successful HIV-1 entry blockade by this C-C chemokine receptor type 5 (CCR5)-antagonist potentiates immunomodulation. We hypothesized that MVC intensification impacts immunization responses, T-cell phenotype, function and delayed type hypersensitivity (DTH) in HIV-1⁺ subjects. A 24-wk, double-blinded, placebo-controlled study of the addition of MVC to suppressive antiretroviral therapy in HIV-1⁺ persons was performed. Subjects received DTH tests, intramuscular tetanus, meningococcal and oral cholera immunizations. Antibody titers, T-cell function and phenotype were assessed. Of 157 patients referred, 47 were randomized 1:1; MVC:placebo. MVC enhanced meningococcal neo-immunization, blunted cholera response and expedited lymphoproliferation to tetanus boost, without affecting recall humoral response. Anti-HIV-1 group-specific antigen (Gag) and tetanus toxoid (TTox) function improved significantly, HIV-1-associated CD8 T-cell skewing normalized, and the percentage of late-stage and major histocompatibility complex (MHC) class II expressing CD4 T-cells increased. Activated CD4⁺ CD38⁺ human leukocyte antigen (HLA)-DR⁺ T-cells declined, and costimulation shifted to coinhibition. DTH was unchanged. Maraviroc intensification, through antagonism of the cell surface molecule CCR5, favorably influences immune profiles of HIV-1⁺ patients, supporting its immunomodulatory use in HIV-1 infection and potentially in other immunologically relevant settings.     

Embryonic stem cells conditioned medium enhances Wharton’s jelly-derived mesenchymal stem cells expansion under hypoxic condition

Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) as compared to typical culture medium for MSCs, Dulbecco’s modified Eagle’s medium with 1.0 g/l glucose (DMEM-LG) supplemented with 10 % FBS, under hypoxic condition. The expanded cells from ESCM (ESCM-MSCs) and DMEM-LG (DMEM-MSCs) were characterized for both phenotype and biological activities including proliferation rate, population doubling time, cell cycle distribution and MSCs characteristics. ESCM and DMEM-LG could enhance WJ-MSCs proliferation as 204.66 ± 10.39 and 113.77 ± 7.89 fold increase at day 12, respectively. ESCM-MSCs could express pluripotency genes including Oct-4, Oct-3/4, Nanog, Klf-4, C-Myc and Sox-2 both in early and late passages whereas the downregulations of Oct-4 and Nanog were detected in late passage cells of DMEM-MSCs. The 2 cell populations also showed common MSCs characteristics including normal cell cycle, fibroblastic morphology, cell surface markers expressions (CD29⁺, CD44⁺, CD90⁺, CD34⁻, CD45⁻) and differentiation capacities into adipogenic, chondrogenic and osteogenic lineages. Moreover, our results revealed that ESCM exhibited as a rich source of several factors which are required for supportive WJ-MSCs proliferation. In conclusion, ESCM under hypoxic condition could accelerate WJ-MSCs expansion while maintaining their pluripotency properties. Our knowledge provide short term and cost-saving in WJ-MSCs expansion which has benefit to overcome insufficient cell numbers for clinical applications by reusing the discarded cell culture supernates from human ES culture system. Moreover, these findings can also apply for stem cell banking, regenerative medicine and pharmacological applications.     

Qualification of a multidonor mixed lymphocyte reaction assay for the functional characterization of immunomodulatory extracellular vesicles

Background aimsExtracellular vesicles (EVs), including exosomes and microvesicles, are released by almost all cells and found in all body fluids. Unknown proportions of EVs transmit specific information from their cells of origin to specific target cells and are key mediators in intercellular communication processes. Depending on their origin, EVs can modulate immune responses, either acting as pro- or anti-inflammatory. With the aim to analyze the immunomodulating activities of EV preparations, especially those from mesenchymal stromal cells (MSCs) in vitro, a multi-donor mixed lymphocyte reaction (mdMLR) assay was established and stressed for its reproducibility.MethodsTo this end, human peripheral blood-derived mononuclear cells (PBMCs) of 12 different healthy donors were pooled warranting mutual allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed PBMCs were cultured for 5 days in the absence or presence of EVs to be tested. Reflecting allogeneic reactions, in the absence of EVs, pooled PBMCs form characteristic satellite colonies whose appearance can be modulated by EVs. More quantifiable, the strength of the allogenic reaction is reflected by the content of activated CD4 and CD8 T cells being recognized by means of their CD25 and CD54 expression.ResultsOf note, connected to the use of primary cells, independent multi-donor PBMC pools differed in their capability to activate their cultured T cells. Thus, throughout the study, only pooled PBMC batches were used whose activated T-cell contents exceeded 25% of the total T-cell population at culture day 5 and whose contents were reproducibly reduced in the presence of immunomodulatory active MSC-EVs. T-cell activation–suppressing effects of the MSC-EV preparations tested were in all cases accompanied by the impact on monocytes. In the presence of immunomodulatory active MSC-EVs, more monocytes were harvested from mdMLR cultures than in their absence. Furthermore, in the absence of immunomodulatory EVs, most monocytes appeared as non-classical (CD14⁺CD16⁺) monocytes, whereas immunomodulatory active MSC-EVs promoted the appearance of classical (CD14⁺⁺CD16^–) and intermediate (CD14⁺⁺CD16⁺) monocyte subpopulations.ConclusionsOverall, the obtained results qualify the mdMLR assay as a robust experimental tool for the evaluation of immunomodulatory potentials of given MSC-EV samples. However, further assay development is required to develop and qualify an authority-acceptable potency assay for clinically applicable MSC-EV products.     

Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells

In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide, extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells in the near future.     

Increased Bone Marrow (BM) Plasma Level of Soluble CD30 and Correlations with BM Plasma Level of Interferon (IFN)-γ, CD4/CD8 T-Cell Ratio and Disease Severity in Aplastic Anemia

Idiopathic aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. Immune abnormalities such as decreased lymphocyte counts, inverted CD4/CD8 T-cell ratio and increased IFN-γ-producing T cells have been found in AA. CD30, a surface protein belonging to the tumor necrosis factor receptor family and releasing from cell surface as a soluble form (sCD30) after activation, marks a subset of activated T cells secreting IFN-γ when exposed to allogeneic antigens. Our study found elevated BM plasma levels of sCD30 in patients with SAA, which were closely correlated with disease severity, including absolute lymphocyte count (ALC) and absolute netrophil count (ANC). We also noted that sCD30 levels were positively correlated with plasma IFN-γ levels and CD4/CD8 T-cell ratio in patients with SAA. In order to explain these phenomena, we stimulated T cells with alloantigen in vitro and found that CD30⁺ T cells were the major source of IFN-γ, and induced CD30⁺ T cells from patients with SAA produced significantly more IFN-γ than that from healthy individuals. In addition, increased proportion of CD8⁺ T cells in AA showed enhanced allogeneic response by the fact that they expressed more CD30 during allogeneic stimulation. sCD30 levels decreased in patients responded to immunosuppressive therapy. In conclusion, elevated BM plasma levels of sCD30 reflected the enhanced CD30⁺ T cell-mediated immune response in SAA. CD30 as a molecular marker that transiently expresses on IFN-γ-producing T cells, may participate in mediating bone marrow failure in AA, which also can facilitate our understanding of AA pathogenesis to identify new therapeutic targets.     

Tolerogenic effect of mesenchymal stromal cells on gliadin-specific T lymphocytes in celiac disease

Background aimsCeliac disease is caused by a dysregulated immune response toward dietary gluten, whose only treatment is a lifelong gluten-free diet. We investigated the effects of mesenchymal stromal cells (MSCs) on gliadin-specific T cells, which are known to induce intestinal lesions, in view of a possible use as new therapy.MethodsBone marrow–derived MSCs and gliadin-specific T-cell lines were obtained from allogeneic donors and mucosal specimens of celiac patients, respectively. The immunosuppressant effect of MSCs was evaluated in terms of proliferative response and interferon (IFN)-γ production upon gliadin stimulation of long-term T-cell lines; the immunomodulant effect was assessed in terms of apoptotic rate, immunophenotype and cytokine profile of short-term T-cell lines generated in the presence of MSCs. Different MSC:T-cell ratios were applied, and statistics were performed as appropriate.ResultsMSCs inhibited both proliferative response and IFN-γ production of long-term T-cell lines in a dose-dependent manner while limiting the expansion of short-term T-cell lines by increasing the apoptotic rate. Moreover, a reduction of the CD4⁺ population and expansion of the regulatory FoxP3⁺ subset were found in T-cell lines cultured with MSCs, in which a significant decrease of interleukin (IL)-21, IFN-γ and IL-10 paralleled by an upregulation of transforming growth factor-β1, IL-6 and IL-8 were observed. Finally, an increase of the indoleamine 2,3-dioxygenase activity was found, possibly playing a key role in mediating these effects.ConclusionsMSCs exert potent immunomodulant effects on gliadin-specific T cells, which may be exploited for future therapeutic application in celiac disease.     

Differential immunomodulation of human mesenchymal stromal cells from various sources in an inflammation mimetic milieu

Mesenchymal stromal cells (MSCs) are very advantageous in the field of regenerative medicine because of their immunomodulatory properties. However, reports show that these properties vary from source to source. Hence, understanding the source-dependent specificity of MSCs and their immunomodulatory abilities will enable optimal use of MSCs in cell-based therapies. Here, we studied human MSCs from three different sources, adipose tissue (AT), bone marrow (BM) and Wharton's jelly (WJ), with respect to phenotypic responses of human peripheral blood mononuclear immune cells (hPBMCs/MNCs) and the concurrent changes in cytokine expression in MSCs, under mitogen-stimulated co-culture conditions. We used cytometric analysis to study the immunoregulatory properties of MSCs on MNCs and cytokine profiling of MSCs using a customized PCR array and solid-phase sandwich enzyme-linked immunosorbent assay. Our results reveal differential modulation of immune cells as well as MSCs upon activation by the mitogen phytohemagglutinin, independently and in co-culture. Notably, we observed source-specific MSC-cytokine signatures under stimulated conditions. Our results show that AT-MSCs up-regulate VEGF, BM-MSCs up-regulate PTGS-2 and WJ-MSCs increase expression of IDO considerably compared with controls. This remarkable modulation in source-specific cytokine expression was also validated at a functional level by quantitative protein expression studies. In our hands, even though MSCs from AT, BM and WJ sources exhibit characteristic immunomodulatory properties, our results highlight that MSCs sourced from different tissues may exhibit unique cytokine signatures and thus may be suitable for specific regenerative applications.     

CD271 enrichment does not help isolating mesenchymal stromal cells from G-CSF-mobilized peripheral blood

Reports on the isolation of mesenchymal stromal cells (MSCs) from granulocyte colony stimulating factor mobilized peripheral blood (G-CSF-mobilized PB) using regular culturing techniques are controversial. Enrichment techniques such as CD133 isolation have increased the success rates. CD271 is a wellknown marker for enrichment of MSCs from bone marrow (BM). In the present study, we aimed to find out whether CD271 enrichment can help isolation of MSCs from G-CSF-mobiiized PB. Five G-CSF-mobilized PB samples were collected from the remnant parts of the bags used for BM transplantation. Five BM samples were used as the control. Mononuclear cells (MNCs) from both resources were collected and underwent magnetic sorting for CD271-positive cells. The isolated cells were cultured, undergoing flowcytometry and differentiation assays to determine if they fulfill MSCs characteristics. CD271-positive portion of G-CSF-mobilized PB did not yield any cell outgrowth but the BM counterpart could successfully form MSC colonies. Although the percentage of CD271⁺ cells showed no difference between BM-MNCs and G-CSF-mobilized PB-MNCs, hematopoietic markers such as CD45, CD34 and CD133 composed a higher percentage of CD271-positive cells in the G-CSF-tnobiiized PB group. Results obtained indicated that CD271 enrichment does not help isolation of MSCs from G-CSF-mobilized PB. In this source, almost all of the CD271⁺ cells are from hematopoietic origin and the frequency of MSCs is so low that possibly during the process of cell isolation most of them are lost and the isolation fails.     

Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression

Indoleamine 2,3-dioxygenase (IDO) functions as a crucial mediator of tumor-mediated immune tolerance by causing T-cell suppression via tryptophan starvation in a tumor environment. Glycogen synthase kinase-3β (GSK-3β) is also involved in immune and anti-tumor responses. However, the relativity of these proteins has not been as well defined. Here, we found that GSK-3β-dependent IDO expression in the dendritic cell (DC) plays a role in anti-tumor activity via the regulation of CD8⁺ T-cell polarization and cytotoxic T lymphocyte activity. By the inhibition of GSK-3β, attenuated IDO expression and impaired JAK1/2-Stat signaling crucial for IDO expression were observed. Protein kinase Cδ (PKCδ) activity and the interaction between JAK1/2 and Stat3, which are important for IDO expression, were also reduced by GSK-3β inhibition. CD8⁺ T-cell proliferation mediated by OVA-pulsed DC was blocked by interferon (IFN)-γ-induced IDO expression via GSK-3β activity. Specific cytotoxic T lymphocyte activity mediated by OVA-pulsed DC against OVA-expressing EG7 thymoma cells but not OVA-nonexpressing EL4 thymoma cells was also attenuated by the expressed IDO via IFN-γ-induced activation of GSK-3β. Furthermore, tumor growth that was suppressed with OVA-pulsed DC vaccination was restored by IDO-expressing DC via IFN-γ-induced activation of GSK-3β in an OVA-expressing murine EG7 thymoma model. Taken together, DC-based immune response mediated by interferon-γ-induced IDO expression via GSK-3β activity not only regulates CD8⁺ T-cell proliferation and cytotoxic T lymphocyte activity but also modulates OVA-pulsed DC vaccination against EG7 thymoma.     

Altered frequency of CD8+CD11c+ T cells and expression of immunosuppressive molecules in lymphoid organs of mouse model of colorectal cancer

CD11c is a member of the β2-integrin family typically used to define myeloid dendritic cells (DCs). Recent reports identify CD11c-expressing CD8⁺ T cells as a new subset of CD8⁺ regulatory T cells (Treg). Evidence exists that CD11c⁺CD8⁺ T cells may exert their effector or regulatory functions under different conditions. To date, no studies have addressed the frequency of CD11c⁺ T cells in cancer. Limited evidence exists in terms of expression of immune-checkpoint receptors, programmed cell death protein 1 (PD-1) and T-lymphocyte-associated antigen 4 (CTLA-4), as well as forkhead box P3 (Foxp3) in mouse lymphoid organs. Here, we have assessed CD11c⁺CD8⁺ and CD11c⁺CD4⁺ T cells, Foxp3, PD-1, and CTLA-4 expressing CD4⁺ T cells and CD8⁺ T cells in different tissues from three groups of male BALB/c mice—young, mature, and those with colorectal cancer (CRC). Analysis of CD3⁺CD11c⁺ T cells in the bone marrow (BM), spleen, and lymph nodes (LN) in each group showed a higher percentage of CD3⁺CD11c⁺ T cells in the BM from all groups and in the lymphoid organs of the cancer group compared with the young and mature groups. CD4^low and CD4^high cell fractions in mice BM have different expression patterns for Foxp3 and CTLA-4. We have observed a higher frequency of CD8⁺PD-1⁺ T cells in the BM, spleen, and LN of CRC mice compared with normal mice. T-cell exhaustion is associated with inhibitory receptor PD-1. According to the regulatory roles of CD11c expression in CD8⁺ T cells, we have proposed that the elevated percentage of CD11c, Foxp3, CTLA-4, and PD-1 expressing T cells were associated with immune response dysregulation in CRC.     

Bone marrow and adipose mesenchymal stem cells attenuate cardiac fibrosis induced by methotrexate in rats

Mesenchymal stem cells (MSCs) are an ideal adult stem cell with capacity for self‐renewal and differentiation with an extensive tissue distribution. The present study evaluates the therapeutic effects of bone marrow mesenchymal stem cells (BM‐MSCs) or adipose‐derived mesenchymal stem cells (AD‐MSCs) against the development of methotrexate (MTX)‐induced cardiac fibrosis versus dexamethasone (DEX). Rats were allocated into five groups; group 1, received normal saline orally; group 2, received MTX (14 mg/kg/week for 2 weeks); groups 3 and 4, treated once with 2 × 10 ⁶ cells of MTX + BM‐MSCs and MTX + AD‐MSCs, respectively; and group 5, MTX + DEX (0.5 mg/kg, for 7 days, P.O.). MTX induced cardiac fibrosis as marked changes in oxidative biomarkers and elevation of triglyceride, cholesterol, aspartate aminotransferase, gamma‐glutamyl transferase, creatine kinase, and caspase‐3, as well as deposited collagen. These injurious effects were antagonized after treatment with MSCs. So, MSCs possessed antioxidant, antiapoptotic, as well antifibrotic effects, which will perhaps initiate them as notable prospective for the treatment of cardiac fibrosis.     

Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia

Multipotent mesenchymal stromal cells (MSCs) from Wharton''s jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.     

Dynamics of Peripheral Blood Lymphocyte Subpopulations in the Acute and Subacute Phase of Legionnaires’ Disease

Study Objective

Absolute lymphocytopenia is recognised as an important hallmark of the immune response to severe infection and observed in patients with Legionnaires’ disease. To explore the immune response, we studied the dynamics of peripheral blood lymphocyte subpopulations in the acute and subacute phase of LD.

Methods and Results

EDTA-anticoagulated blood was obtained from eight patients on the day the diagnosis was made through detection of L. pneumophila serogroup 1 antigen in urine. A second blood sample was obtained in the subacute phase. Multiparametric flow cytometry was used to calculate lymphocyte counts and values for B-cells, T-cells, NK cells, CD4⁺ and CD8⁺ T-cells. Expression of activation markers was analysed. The values obtained in the subacute phase were compared with an age and gender matched control group. Absolute lymphocyte count (×10⁹/l, median and range) significantly increased from 0.8 (0.4–1.6) in the acute phase to 1.4 (0.8–3.4) in the subacute phase. B-cell count showed no significant change, while T-cell count (×10⁶/l, median and range) significantly increased in the subacute phase (495 (182–1024) versus 979 (507–2708), p = 0.012) as a result of significant increases in both CD4⁺ and CD8⁺ T-cell counts (374 (146–629) versus 763 (400–1507), p = 0.012 and 119 (29–328) versus 224 (107–862), p = 0.012). In the subacute phase of LD, significant increases were observed in absolute counts of activated CD4⁺ T-cells, naïve CD4⁺ T-cells and memory CD4⁺ T-cells. In the CD8⁺ T-cell compartment, activated CD8⁺ T-cells, naïve CD8⁺ T-cell and memory CD8⁺ T-cells were significantly increased (p<0.05).

Conclusion

The acute phase of LD is characterized by absolute lymphocytopenia, which recovers in the subacute phase with an increase in absolute T-cells and re-emergence of activated CD4⁺ and CD8⁺ T cells. These observations are in line with the suggested role for T-cell activation in the immune response to LD.     

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.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

A Trifunctional Dextran-Based Nanovaccine Targets and Activates Murine Dendritic Cells,and Induces Potent Cellular and Humoral Immune Responses In Vivo

Dendritic cells (DCs) constitute an attractive target for specific delivery of nanovaccines for immunotherapeutic applications. Here we tested nano-sized dextran (DEX) particles to serve as a DC-addressing nanocarrier platform. Non-functionalized DEX particles had no immunomodulatory effect on bone marrow (BM)-derived murine DCs in vitro. However, when adsorbed with ovalbumine (OVA), DEX particles were efficiently engulfed by BM-DCs in a mannose receptor-dependent manner. A DEX-based nanovaccine containing OVA and lipopolysaccharide (LPS) as a DC stimulus induced strong OVA peptide-specific CD4⁺ and CD8⁺ T cell proliferation both in vitro and upon systemic application in mice, as well as a robust OVA-specific humoral immune response (IgG1>IgG2a) in vivo. Accordingly, this nanovaccine also raised both a more pronounced delayed-type hypersensitivity response and a stronger induction of cytotoxic CD8⁺ T cells than obtained upon administration of OVA and LPS in soluble form. Therefore, DEX-based nanoparticles constitute a potent, versatile and easy to prepare nanovaccine platform for immunotherapeutic approaches.