Advertisement

Immunomodulation by placenta-derived decidua stromal cells. Role of histocompatibility, accessory cells and freeze–thawing

  • Behnam Sadeghi
    Correspondence
    Correspondence: Behnam Sadeghi, MD, PhD, Associate Professor, Translational Cell Therapy Research (TCR), Kliniskt Forskningscentrum (KFC), Novum, Hälsovägen 7-9, 141 57 Huddinge, Sweden.
    Affiliations
    Translational Cell Therapy Research (TCR), Division of Pediatrics, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
    Search for articles by this author
  • Myrèse Witkamp
    Affiliations
    Translational Cell Therapy Research (TCR), Division of Pediatrics, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
    Search for articles by this author
  • Dominik Schefberger
    Affiliations
    Translational Cell Therapy Research (TCR), Division of Pediatrics, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
    Search for articles by this author
  • Anna Arbman
    Affiliations
    Translational Cell Therapy Research (TCR), Division of Pediatrics, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
    Search for articles by this author
  • Olle Ringdén
    Affiliations
    Translational Cell Therapy Research (TCR), Division of Pediatrics, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Huddinge, Sweden
    Search for articles by this author
Open AccessPublished:November 01, 2022DOI:https://doi.org/10.1016/j.jcyt.2022.10.004

      Abstract

      Background aims

      Human placenta-derived decidua stromal cells (DSCs) are newly introduced stromal cells that have successfully been used in several clinical trials for the treatment of acute inflammatory diseases. Despite published data about DSCs, deeper exploration of mechanisms of action and crosstalk with other immune cells need to be explored.

      Methods

      In mixed lymphocyte culture (MLC), the splenocytes from Balb/c or B6 mice were stimulated using mitogen (concanavalin A), allogeneic (B6 or Balb/c splenocytes) or xenogeneic activation with human peripheral blood mononuclear cells.

      Results

      When 10% of the mouse bone marrow-derived–MSC, being autologous, allogeneic or haploidentical (from F1), was added, >95% inhibition was seen. Using human (h)-DSCs, the inhibitory capacity was a median 68% as a xenogeneic immunomodulatory cell when used in mitogen and allogeneic setting in mice MLC. However, when human peripheral blood mononuclear cells were used as stimulator for mouse splenocyte (xenogeneic MLC), hDSC showed a median inhibition of 88%. We explored the presence and function of monocytes in the immunomodulatory function of stromal cells. CD14+ monocyte cells reduced the immunosuppressive effect by hDSC. hDSCs did not show any inhibitory effect on natural killer cell activation and proliferation by interleukin-2. In contrast DSCs increased natural killer proliferation by a median of 58%. Fresh or frozen–thawed hDSCs had similar inhibitory effects on human T-cell proliferation (both allo-stimulation and mitogen stimulation) in vitro. Cell viability at room temperature during 24 h was similar using fresh or freeze–thawed DSCs.

      Conclusions

      To conclude, histocompatibility and CD14+ monocyte cells had an impact on hDSC immunomodulation but frozen–thawed or freshly prepared cells did not.

      Key Words

      Introduction

      Human mesenchymal stromal cells (hMSCs) are very rare precursors in all tissues of the body [
      • Friedenstein A.J.
      • Chailakhjan R.K.
      • Lalykina K.S.
      The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells.
      ,
      • Campagnoli C.
      • et al.
      Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow.
      ,
      • Moll G.
      • et al.
      Intravascular Mesenchymal Stromal/Stem Cell Therapy Product Diversification: Time for New Clinical Guidelines.
      ]. MSCs have immunosuppressive properties and are therefore used for the treatment of inflammatory disorders such as graft-versus-host disease (GVHD), acute respiratory distress syndrome, multiple sclerosis, rheumatic diseases, etc. [
      • Ringden O.
      • et al.
      Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease.
      ,
      • Hashmi S.
      • et al.
      Survival after mesenchymal stromal cell therapy in steroid-refractory acute graft-versus-host disease: systematic review and meta-analysis.
      ,
      • Zheng G.
      • et al.
      Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study.
      ,
      • Sadeghi B.
      • et al.
      Conquering the cytokine storm in COVID-19-induced ARDS using placenta-derived decidua stromal cells.
      ,
      • Wang Y.
      • et al.
      Clinical application of mesenchymal stem cells in rheumatic diseases.
      ,
      • Zhuang X.
      • et al.
      Mesenchymal Stem Cell-Based Therapy as a New Approach for the Treatment of Systemic Sclerosis.
      ,
      • Ringden O.
      • et al.
      Mesenchymal Stromal Cells for Enhancing Hematopoietic Engraftment and Treatment of Graft-Versus-Host Disease, Hemorrhages and Acute Respiratory Distress Syndrome.
      ]. hMSCs from all tissues and even fibroblasts have immunosuppressive capacities [
      • Bartholomew A.
      • et al.
      Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.
      ,
      • Karlsson H.
      • et al.
      Stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro.
      ,
      • Jones S.
      • et al.
      The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells.
      ].
      The fetus is protected from the mother's human leukocyte antigen (HLA)-incompatible immune system by the fetal membrane and the placenta [
      • Mincheva-Nilsson L.
      Immune cells and molecules in pregnancy: friends or foes to the fetus?.
      ]. Human placenta–derived, so-called, decidua stromal cells (hDSCs), provide stronger immunosuppression in vitro and in vivo compared with human MSCs from bone marrow (BM) or other tissues [
      • Karlsson H.
      • et al.
      Stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro.
      ,
      • Sadeghi B.
      • et al.
      Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo.
      ,
      • Ringden O.
      • et al.
      Placenta-Derived Decidua Stromal Cells for Treatment of Severe Acute Graft-Versus-Host Disease.
      ]. hDSCs have similar positive/negative surface markers like all sources of hMSCs [
      • Ringden O.
      • et al.
      Fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease.
      ]. Apart from the stronger immunosuppressive effects of hDSCs, there are also several other differences compared with hBM-MSCs. hDSCs are only half the size of hBM-MSCs and, in addition, they do not differentiate as well to bone and fat [
      • Erkers T.
      • et al.
      Decidual stromal cells promote regulatory T cells and suppress alloreactivity in a cell contact-dependent manner.
      ,
      • Moll G.
      • et al.
      Different Procoagulant Activity of Therapeutic Mesenchymal Stromal Cells Derived from Bone Marrow and Placental Decidua.
      ,
      • Kazemi S.
      • et al.
      Growth kinetic comparison of Human Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue and Decidua.
      ]. Furthermore, hDSCs inhibit human and mouse T-cell proliferation in vitro in a contact-dependent manner and not mainly by soluble factors as do hBM-MSCs. Blocking experiments suggest that interferon-γ, prostaglandin E, indoleamine-2, 3-dioxygenase (IDO) and programmed death-ligand 1 (PD-L1) are involved in the immunosuppressive mechanisms of DSCs [
      • Erkers T.
      • et al.
      Decidual stromal cells promote regulatory T cells and suppress alloreactivity in a cell contact-dependent manner.
      ].
      Immune response involves a complicated multicellular crosstalk including several accessory cells. Macrophages and other types of accessory cells seem to be important in the immunosuppression induced by hMSCs [
      • Parekkadan B.
      • et al.
      Bone marrow stromal cell transplants prevent experimental enterocolitis and require host CD11b+ splenocytes.
      ,
      • Galleu A.
      • et al.
      Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.
      ]. In the current report, we present our findings regarding the effect of monocyte in the immunomodulatory capacity of hDSCs.
      In the clinical cell therapy setting, an important issue is the advantage/disadvantage of freeze–thawed versus freshly prepared cells for clinical practice. The freeze–thawing process is reported to negatively affect MSCs, their coagulability and immunological effects [
      • Hoogduijn M.J.
      • et al.
      Effects of Freeze-Thawing and Intravenous Infusion on Mesenchymal Stromal Cell Gene Expression.
      ,
      • Chinnadurai R.
      • et al.
      Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.
      ,
      • Moll G.
      • et al.
      Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?.
      ,
      • Moll G.
      • Hoogduijn M.J.
      • Ankrum J.A.
      Editorial: Safety, Efficacy and Mechanisms of Action of Mesenchymal Stem Cell Therapies.
      ]. We evaluated and compared these two preparation methods in immunosuppressive capacity as well as cell viability.
      Although we have a lot of information about the mechanisms of action as well as crosstalk between stromal cells and different immune cells [
      • Karlsson H.
      • et al.
      Stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro.
      ,
      • Sadeghi B.
      • et al.
      Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo.
      ,
      • Ringden O.
      • et al.
      Fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease.
      ,
      • Erkers T.
      • et al.
      Decidual stromal cells promote regulatory T cells and suppress alloreactivity in a cell contact-dependent manner.
      ], but still there are several unidentified subjects in our knowledge about these cells. Resolving these issues may tune and improve stromal cells efficacy in clinical setting. In this communication, we studied hDSCs and their immunomodulatory effects with special reference to the roles of the histocompatibility system, accessory cells and using frozen–thawed DSCs versus fresh DSCs isolated from the expansion culture.

      Material and methods

      Preparation of hDSCs

      Isolation, preparation and expansion of hDSCs was published previously in detail [
      • Ringden O.
      • et al.
      Fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease.
      ]. In short, the placentas were obtained from healthy mothers after signing the informed consent and following elective cesarean delivery. The fetal membrane was dissected carefully from the placenta. Subsequently, it was digested with trypsin/ethylenediaminetetraacetic acid (EDTA; Thermo Fisher Scientific, Waltham, MA, USA) using a series of incubations and washes. Trypsin-digested cell suspensions or tissue were washed and seeded in Nunc T175 flasks (Nunc A/S) using complete Dulbecco's modified Eagle's medium (cDMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone; Thermo Fisher Scientific). When the cells were approximately 90–95% confluent, they were harvested with trypsin/EDTA, washed with cDMEM and fed in new T175 flasks at 2.9 × 103 cells/cm2. The adherent cells were cultured to 2, 3 or more times (up to P5) and frozen slowly in cDMEM containing 10% dimethyl sulfoxide (Wak-Chemie Medical Gmbh, Steinbach, Germany). hDSCs were studied and cultured under good laboratory practice conditions using a room with reverse isolation, a sterile bench and a separate incubation for cells from each donor. For this study, we used DSCs from four different donor (Batch)

      Isolation and preparation of mouse (m) BM-MSC

      Femur and tibias from 6- to 8-week-old Balb/c, B6 and their F1 generation (Balb/c x B6) mice were dissected. Crude BM was collected by cutting up the bone and inserting 18-gauge syringe at the bone cavity and washing it with 5 mL of phosphate-buffered saline (HyClone) containing 10% FBS. Isolated cells were centrifuged at 1200 rpm for 10 minutes at room temperature. Cell pellets (crude BM cells) were resuspended in 8 mL of alpha-Minimum Essential Medium (Gibco/Thermo Fisher Scientific) supplemented with 10% FBS (HyClone) supplemented with 100 μg/mL penicillin/100 μg/mL streptomycin. Cells were seeded in T75 flask and placed in CO2 incubator with 95% humidity for several days. At 90% confluence, the adherent cells were washed with phosphate-buffered saline and detached from the flask using trypsin/EDTA. Isolated cells were sub-cultured for at least four passages (P4). In all experiments we used mBM-MSC at passage 5 or more when cell surface markers showed MSCs characteristics. Cell counting was performed in a Bruckner counting chamber using Trypan Blue solution.

      Human or mouse mononuclear cells preparations

      Human peripheral blood mononuclear cells (hPBMCs) were isolated from the buffy coats of the blood donated by healthy volunteer using density gradient centrifugation (Ficoll separation method) (Lymphoprep; Axis, Oslo, Norway). Mouse splenocytes were obtained from mechanical disaggregation of freshly harvested spleens from mice killed humanly and under the hood (sterile environment). After several washings and a centrifugation process, red blood cells were lysed and clean mononuclear cells were collected. In the experiments, lymphocytes from at least three different individuals were used. Isolated cells were frozen until each relevant experiment.

      Immune phenotyping of mBM-MSC

      The phenotype of the mBM-MSCs was characterized by flow cytometry analysis using BD Accuri C6 plus (BD Biosciences, San José, CA, USA). For cell flow cytometric analyses, Accuri software was used to analyze the acquired data. Different mBM-MSC, from Balb/c, B6 or F1 (Balb/c x B6) were stained with the most popular positive/negative markers used to characterize mBM-MSCs. Fluorescein isothiocyanate–positive antibodies were used for CD44, CD45, CD29 and CD90.2 and phycoerythrin-positive antibodies were used for CD11b, CD34, CD105, CD117 (C.kit) and Sca-1 cell-surface markers (BD Biosciences, San Diego, CA, USA).

      Mixed lymphocyte cultures (MLCs)

      MLC is an in vitro test. In MLC, human or animal lymphocytes (responder cells) are stimulated by inactivated allogeneic, xenogeneic cells. PBMCs also were activated by mitogens, e.g., Concavalin A (Con A) or phytohemagglutinin (PHA). Responder lymphocytes secrete cytokines and proliferate in the culture. After certain number of days, the proliferation rate is measured (and compared with controls) via several methods, including 3H-thymidine, which is a radioisotope and incorporated in replicating DNA. 3H-Thymidine demonstrates the cell proliferation ratio in different wells. Adding efficient immunomodulatory cells/-pharmacologic agents into the experimental assays prevents proliferation of responder lymphocytes and accordingly reflects the power of immunomodulator/immunosuppressor agents.
      Responder cells, mouse splenocytes (from Balb/c or B6 mice) or hPBMCs, 4 × 105 cells/well were co-cultured with 2 × 105 irradiated (30 Gy) splenocytes (from B6 or Balb/c mice, respectively) or 1 × 105 irradiated hPBMCs as stimulators with or without hDSCs or various type of mBM-MSCs in different ratios in 96-well plates (0.2 mL/well; round bottom; Corning Costar, Corning, NY, USA). Pooled hPBMCs (stimulator) from five different donors were prepared by mixing PBMCs from at least five different donors to ensure variability at the HLA-D loci.
      After 3 or 5 days, the cultures were pulsed during the final 18 hours with 1 μCi/well 3H thymidine (Perkin-Elmer, Waltham, MA, USA). The cells were harvested from a Harvester 96 (Tomtec, Munich, Germany) and β radiation was measured using a Trilux 1450 MicroBeta microplate scintillation counter (Wallac Sweden AB). If not otherwise stated, the ratio of mMSCs or hDSCs compared to responder cells was 10%.

      Transwell proliferation assay

      Transwell experiments were performed to examine whether direct contact between T cells and hDSCs or the production of soluble factors are important for the immunosuppressive function. The MLC co-culture cells were separated from hDSCs by a 1-μm pore size membrane (1-μm pore size WWR, Radnor, PA, USA). MLC cells were seeded in the lower chamber whereas hDSCs were seeded in the upper chamber. Transwell experiments were performed in 12-well plates (concentration: 2 mL/well), whereas regular MLCs were performed in triplicate in 96-well plates (concentration: 0.2 mL/well). MLCs containing CD3+ T cells stimulated with CD28 were incubated for 3 days in a 37°C, 5% CO2 incubator whereas MLCs containing CD56+ natural killer (NK) cells were incubated for 6 days.

      Preparation of T cells, NK cells and monocytes

      CD3+ T cells, CD14+ monocytes and CD56+ NK cells were isolated from PBMC by positive selection using CD3, CD14 and CD56 microbeads (Miltenyi Biotec, Bergisch, Gladbach, Germany) following the manufacturer's instructions. PBMCs were labeled by magnetic CD3, CD14 or CD56 microbeads and were incubated for 15 min. The cells of interest were isolated using a magnetic column (Miltenyi Biotec). Not interested cells were washed away. To increase the purity of the cells in some cases, the magnetic preparation was repeated twice.

      Frozen–thawed versus freshly prepared hDSCs

      We examined five different batches of frozen hDSC from different years. When the cells were thawed, one part was resuspended in the infusion buffer. Cells were resuspended in sodium chloride 0.9% supplemented with 5% human serum albumin. Cell viability was measured, and cell numbers and viability were counted at starting point (T0), 0.5, 1, 3, 6, 18 and 24 h after thawing. The cells were kept in room temperature (21°C) during the experiment time (24 h). This group is referred as frozen–thawed cells. The other half of the cells, after thawing, were seeded and cultured. After confluency of more than 85%, culture flasks were trypsinized and hDSCs were washed and collected and resuspended in the same infusion buffer. Cell viability and cell numbers were measured in the similar condition as frozen cells. This group were considered as fresh cells.

      Statistical analysis

      Comparisons between two groups were performed using the independent-samples t-test. A value of P < 0.05 was considered statistically significant. SPSS statistics, version 22 (IBM Corp., Armonk, NY, USA) was used to conduct the statistical analysis and Graph Pad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used to create graphs. Box plots were constructed with error bars showing the 2.5 and 97.5 percentiles, and the mean was indicated by a black line. The notations used for significance were as follows: *P < 0.05, ** P < 0.01, ***P < 0.001 and ****P < 0.0001.

      Results

      Characterization of and phenotype analysis of mouse BM-MSCs

      mMSCs were isolated and expanded from the BM cells of Balb/c, B6 or F1 (Balb/c x B6) mice. Usually myeloid and lymphoid contamination vanish after passage 4. Therefore, in all experiments we used mMSCs from passage 5 or greater. Figure 1,A shows the spindle-shaped morphology of mMSCs from three different strains. Among them, F1 had better proliferation capacity and less doubling time. Flow cytometric analyses revealed some differences in expression level of cell surface markers between Balb/c, B6 and F1 mice. mMSCs from Balb/c, B6 and F1 (Balb/c x B6) at passage 5 were analyzed for cell surface markers. All positive and negative markers in a typical histogram are shown in Figure 1,B. As expected, stromal cells from Balb/c, B6 and F1 mice showed different expression levels in CD34, CD44, CD73 and CD105. However, they similarly were positive for CD29 and Sca-1 and negative for CD11b and CD45 (Figure 1,B).
      Figure 1
      Figure 1Phenotypic analysis of DSCs by flow cytometry. The colored graphs show the expression of the different cell surface markers (filled areas under the curve) compared with unstained DSC controls (white areas under the curve). The percentages represent the size of each population as a proportion of the whole population. MSCs from all mice strains originated from BM.

      The effect of histocompatibility of stromal cell and immunomodulatory function

      Splenocytes from Balb/c mice were stimulated with Con A (Figure 2). When autologous (from Balb/c), allogeneic (from B6) or major histocompatibility complexes (MHCs) haploidentical (from F1) MSCs were added, a strong inhibitory effect on proliferation was seen (Figure 2,A). Median inhibitory effect of proliferation was 98.5% (P < 0.02, N = 4), 98.9% (P < 0.02, N = 4) and 98.8% (P < 0.02, N = 4) in the three groups, respectively. When hDCS was added to the Con A stimulated Balb/c splenocytes, the inhibition was a median of 49.7%, although the inhibition was less, compared with mouse MSCs, but it was a significant decrement in the splenocyte proliferation (P < 0.02, N = 4). In an alloreactive MLC setting, where Balb/c splenocytes were stimulated with mitomycin-treated B6 splenocytes, autologous, allogeneic and haploidentical BM-MSCs all showed almost complete inhibition of proliferation (Figure 2,B). Autologous mBM-MSCs showed a median inhibition of 98.2%. For the allogeneic mMSCs, the median inhibition was 88.7% and for the haploidentical MSCs, the median inhibition was 95.7% from two to three different experiments. In this animal alloreactive MLC setting, the hDSCs showed a median inhibition of the mouse´s MLC of 53.6% (Figure 2,B). Due to the sample size (N = 2 or 3) and variance in medians we could not show statistically significant differences.
      Figure 2
      Figure 2Effect of MHCs on stromal cell function. Splenocyte from Balb/c (A-C) or B6 (D-F) mice, as responder or stimulator, were stimulated with (A,D) mitogenic (Con A), (B,E) allogeneic (Balb/c as responder and irradiated B6 as stimulator or vice versa) or (C,F) xenogeneic stimulators (Balb/c or B6 splenocyte as responder and irradiated human lymphocyte as stimulator). (A) Mitogenic stimulated Balb/c splenocytes (responder) were suppressed by adding mBM-MSCs from autologous (Balb/c), allogeneic (B6), haploidentical (F1) and xenogeneic (hDSCs). hDSCs were not able to suppress proliferation as good as other stromal cells. (B) Balb/c splenocytes (responder) were stimulated by irradiated splenocyte from B6 mice (allogeneic stimulator) and were then suppressed by adding mBM-MSCs from autologous (Balb/c), allogeneic (B6), haploidentical (F1) and xenogeneic (hDSCs). In this setting hDSCs were not able to suppress proliferation as good as other stromal cells. (C) Balb/c splenocytes (responder) were stimulated by irradiated human PBMCs (xenogeneic stimulator) and then were suppressed by adding mBM-MSCs from autologous (Balb/c), allogeneic (B6), haploidentical (F1) and xenogeneic (hDSCs). Opposite to other set of MLCs, hDSCs could strongly suppress MLC when stimulator was human PBMCs. (D), (E), and (F) are like (A), (B) and (C) but the responder cells are from B6 mice. Similar observation. Mice MSCs used in these experiments all originated from BM.
      Balb/c splenocytes were stimulated with irradiated hPBL (human peripheral blood lymphocytes) to study xenoreactivity (Figure 2,C). In this experiment, there was a strong suppressive effect by autologous, allogeneic and haploidentical BM-MSCs: 98.2%, 98.6 and 98.2% in the three groups, respectively. Interestingly, the inhibitory effect by hDSCs was very strong, median 90.6% (Figure 2,C), compared with using hDSCs in mitogenic, median 49.7% inhibition (Figure 2,A), or allogeneic MLC, where median inhibition was 53.6% (Figure 2,B).
      In a similar set of experiments, splenocytes from B6 mice were used as responder and were stimulated by Con A (Figure 2,D). Using autologous mBM-MSCs (from B6), the median inhibitory effect from four experiments was 70.7% (P < 0.02, N = 4). For allogeneic mBM-MSCs (from Balb/c) the median inhibition was 63.1% (P < 0.02, N = 4) and for the haploidentical mBM-MSCs (from F1) it was 81% (P < 0.02, N = 4). Using xenogeneic hDSC the median inhibition of B6 splenocytes, stimulated with Con A, was median 52.5% (P < 0.02, N = 4). In an allogeneic MLC setting, the B6 splenocytes were stimulated with Balb/c mice splenocytes (mitomycin-treated) and a strong inhibition of proliferation was seen using mBM-MSCs (Figure 2,E). The median inhibition in the MLC was 98.9% using autologous mBM-MSCs and 98.5% using allogeneic mBM-MSCs in three different experiments. Using haploidentical mBM-MSCs the median inhibition was 98.8%. In the same setting of MLC where hDSCs were added to the culture, the median inhibition was 49.7% (Figure 2,E). However, when irradiated human pooled PBMC were used as stimulator (Xeno-MLC) the median inhibition by hDSCs was 63.2% (Figure 2,F) (P < 0.02, N = 4), whereas autologous (from B6) (P < 0.02, N = 4), allogeneic (from Balb/c) (P < 0.02, N = 4) and haploidentical (from F1) (P < 0.02, N = 4) mBM-MSC showed strong and similar inhibition on B6 responder cells (Figure 2,F).

      DSCs works in Transwell when stimulated by responder T cells

      hDSCs suppress CD3/CD28 stimulated T cells when added in culture when there is direct contact (Figure 3,A, the first two Bar-Box from the left, P < 0.0001, N = 5). When T cells and hDSCs were separated by the insert in Transwell culture setting, the hDSCs did not inhibit T-cell proliferation (Figure 3,A, first and third bar-boxes from the left; P < 0.0001, N = 5). Interestingly, when T cells were added over the insert and hDSCs were in direct contact with limited number of T cells, they could secrete some mediators, which could suppress the proliferation of stimulated T cells in lower chamber, where they did not have any cell-to-cell contact with hDSCs (Figure 3,A, first and fourth bar-boxes from the left, P < 0.02, N = 4).
      Figure 3
      Figure 3Effect of cell-to-cell contact and role of accessory cells in DSC function. (A) T cells were stimulated in the lower chamber (Down) using CD3/CD28 Abs (A, first left column, control). DSCs were cocultured with T cells (Dir) (A, second left column, direct cell to cell contact) or seeded over (Up) the insert (1 μm) (A, third left column, no cell-to-cell contact). When DSCs did not have direct contact T cells, they couldn't suppress proliferation. Adding 105 of T cells from the same donor over the insert (A, fourth left column, cell-to-cell contact, added T cells make cell-to-cell contact with DSCs) resulted inhibition of T-cell proliferation in the lower chamber (in which did not face any DSCs). Adding monocytes over the insert didn't have the same effect. In this figure / means the insert! (B) T cells were stimulated using CD3/CD28 Abs (B, first left column, control). Adding DSCs to the T cells significantly decreased T-cell proliferation (B, second left column). Adding back the autologous monocytes (same as T cells) decreased inhibitory effects of DSCs (B, third left column). Adding back monocytes to the MLC did not change T-cell proliferation (B, fourth left column). (C) NK cells were stimulated with IL-2 (C, first left column, control). When hDSCs were added to the control wells (NK cell + IL-2), NK-cell proliferation was even more increased (C, second left column). Adding pooled PBMCs (as an allogeneic stimulator for NK) to the stimulated NK cells (+IL-2) increased NK- cell proliferation (C, third left column) while adding hDSCs did not influence NK activity against PBMCs (C, first right column).

      Effects of CD14+ monocytes on immunomodulatory function of DSCs

      CD14+ monocytes/macrophages play essential roles in immunoregulation [
      • Mills C.D.
      • et al.
      M-1/M-2 macrophages and the Th1/Th2 paradigm.
      ]. Due to the immunomodulatory properties of CD14+ cells, we wanted to analyze CD14+ cell effects on hDSCs capacity to inhibit activated CD3+ T cells. CD14+ monocytes/macrophages reduced the immunosuppressive effects of hDSCs (Figure 3,B). In eight independent experiments, the hDSCs from two different donors were added to activated enriched CD3+ T cells in the absence or presence of irradiated autologous CD14+ cells. CD14+ cells from the same individuals (responder cells) mostly reduced the suppressive effect of hDSCs on activated T cells (Figure 3,B, second and third bar-boxes from the left, P < 0.0001, N = 8). The addition of autologous CD14+ cells did not affect the proliferation of activated autologous T cells (data not shown).

      Human DSCs effects on NK-cell proliferation

      We aimed to investigate the immunomodulatory effects of hDSCs on NK cells. NK cells were activated using interleukin 2 (IL-2) and cultured in the presence or absence of hDSCs. NK-cell proliferation status was determined 3 and 6 days after co-culture. NK-cell proliferation was significantly increased by 41% when hDSCs were added to the culture (P < 0.0001, N = 4; Figure 3,C). Thereafter, we wanted to investigate the effects of hDSCs on NK proliferation in an allogeneic setting. Irradiated pooled allogeneic PBLs were added to NK cells stimulated with IL-2 (Figure 3,C, N = 4). NK cell proliferation was increased by 54% (P < 0.0001). When hDSCs were added, NK proliferation was not affected using two different batches of hDSCs (Figure 3,C, N = 4).

      Immunomodulation capacity in fresh versus frozen–thawed DSCs

      Human PBMCs were stimulated by PHA (Figure 4,A), and the hPBMC proliferation was inhibited by adding either fresh or frozen–thawed hDSCs to the similar extent, 54% (P < 0.0001, N = 4) and 69% (P < 0.0001, N = 4), respectively. Fresh and frozen–thawed hDSCs inhibited proliferation, which was slightly stronger for the frozen–thawed hDSCs (Figure 4,A, P < 0.04, N = 6). In another setting, hPBMCs were stimulated with irradiated pooled hPBMCs and were incubated for 5 days (Figure 4,B). The proliferation significantly decreased by both fresh (P < 0.0001, N = 3) and frozen–thawed (P < 0.0001, N = 3) hDSCs. There were not significant differences in the suppressive capacity of fresh or frozen–thawed hDSCs (Figure 4,B, N = 3).
      Figure 4
      Figure 4Fresh versus frozen–thawed DSCs function. (A) PBMCs were stimulated with PHA, adding 10% hDSCs either frozen–thawed or fresh (from the unique donor) suppressed the proliferation of PBMCs. However frozen–thawed cells performed significantly better. (B) PBMCs were stimulated with irradiated pooled PBMC's, adding 10% hDSCs either frozen–thawed or fresh (from the same donor) strongly suppressed the proliferation of PBMCs. There were no differences in suppressive power between frozen–thawed or fresh cells. (C) Cell viability and (D) absolute number of frozen–thawed or freshly prepared hDSCs when measured during 24 h in room temperature. There was no significant drop in cell viability or number if cell vehicle and preparation was done properly.
      Cell viability and cell counts of fresh and frozen–thawed hDSCs did not change from time 0 up to 24 h after harvesting/thawing the cells and keeping them in infusion buffer in room temperature (Figure 4,C). Whatever the viability was at the beginning (at 0 hours), it was kept until 24 hours both for fresh and frozen–thawed hDSC. For comparison, one batch of DSCs had less than 50% viability at start. The viability remained on the same level for 24 hours (Figure 4,C). In the same batch after expansion and re-culturing the cells, they refreshed in flasks and showed and kept the viability >90% up to 24 hours. An important issue in this assay setting, which should be considered, is the absolute number of the cells. We evaluated the absolute number of fresh and frozen–thawed DSCs up to 24 hours. As shown in Figure 4,D total number of cells was not affected in room temperature up to 24 hours (Figure 4,D).

      Discussion

      In the first clinical trial using hMSCs, autologous cells were co-infused in patients treated with autologous hematopoietic cell transplantation (HCT) for breast cancer [
      • Koç O.N.
      • et al.
      Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.
      ]. In a subsequent study in HLA-identical siblings, BM-MSCs were co-infused at the time of transplant in allogeneic HCT [
      • Lazarus H.M.
      • et al.
      Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.
      ]. In the latter study, MSCs and HCT were from the same HLA-identical donor. We found that BM-MSCs were immunosuppressive in MLC regardless of HLA-compatibility between MSCs and responder or stimulator lymphocytes [
      • Le Blanc K.
      • et al.
      Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.
      ]. These findings paved the way for using third-party haploidentical BM-MSCs for life-threatening acute GVHD [
      • Le Blanc K.
      • et al.
      Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.
      ]. In a subsequent multicenter study using BM-MSCs from various donors in the treatment of steroid-refractory acute GVHD, we found that the outcome was similar using HLA-identical, haplo-identical or HLA-mismatched BM-MSCs for therapy [
      • Le Blanc K.
      • et al.
      Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.
      ]. Following this, third party MSCs were used in a prospective randomized trial [
      • Martin P.J.
      • et al.
      Prochymal Improves Response Rates In Patients With Steroid-Refractory Acute Graft Versus Host Disease (SR-GVHD) Involving The Liver And Gut: Results Of A Randomized, Placebo-Controlled, Multicenter Phase III Trial In GVHD.
      ,
      • Kebriaei P.
      • et al.
      A Phase 3 Randomized Study of Remestemcel-L versus Placebo Added to Second-Line Therapy in Patients with Steroid-Refractory Acute Graft-versus-Host Disease.
      ].
      It is important to investigate the role of the major histocompatibility complex for the immunosuppressive capacity of stromal cells including hDSCs. However, the experience using hDSCs is more limited than BM-MSCs. In the initial animal studies, we found that hDSCs had a xeno-immunosuppressive effect on mouse splenocyte proliferation, stimulated in allogeneic, xenogeneic or mitogenic settings [
      • Sadeghi B.
      • et al.
      Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo.
      ]. In the clinical application of hDSCs, third-party cells were used regardless of HLA compatibility between hDSC donors and recipient [
      • Ringden O.
      • et al.
      Placenta-Derived Decidua Stromal Cells for Treatment of Severe Acute Graft-Versus-Host Disease.
      ,
      • Ringden O.
      • et al.
      Fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease.
      ,
      • Aronsson-Kurttila W.
      • et al.
      Placenta-Derived Decidua Stromal Cells for Hemorrhagic Cystitis after Stem Cell Transplantation.
      ]. By using HLA-mismatched DSCs or other types of stromal cells, there is a risk of immunization and formation of alloreactive multi-specific anti-HLA antibodies [
      • Kaipe H.
      • et al.
      Immunogenicity of decidual stromal cells in an epidermolysis bullosa patient and in allogeneic hematopoietic stem cell transplantation patients.
      ]. Moreover, it is crucial to explore the role of HLA disparity between the stromal cells versus recipient immune cells and immunosuppressive outcome.
      The present analysis showed that autologous, MHC haplo-identical or MHC completely mismatched BM-MSCs had the same immunosuppressive capacity regarding mitogenic, allogeneic and xenogeneic stimulation (Figure 2). However, xenogeneic immunomodulatory cells, hDSCs, had a lower immunosuppressive effect. In human settings, it is not practically possible to evaluate the effect of hDSCs comparing autologous versus various HLA-mismatched donors. Therefore, we must rely on the data from mouse models. In addition, we used two different strains of mice, BALB/c and B6, which differ in their immune systems [
      • Watanabe H.
      • et al.
      Innate immune response in Th1- and Th2-dominant mouse strains.
      ]. B6 and BALB/c differ in their innate immune responses, primarily between type 1 and type 2 helper T cells (Th1 and Th2). This may explain the finding that lymphocytes from B6 mice did not respond as strong as lymphocytes from Balb/c mice and there were differences in activation patterns by mitogens and in the MLR.
      In the xeno-MLC when the Balb/c lymphocytes (responder cells) were stimulated with pooled human PBMCs (as stimulator), adding hDSCs showed stronger suppression as compared with the allogeneic-MLC setting, where B6 splenocyte (allogeneic cells) were used as stimulators (Figure 2B,C). This may suggest that analogous human MHC molecules on stimulator cells and hDSCs are of importance for suppression to occur.
      Lack of immunization may be an advantage when using autologous hMSCs in regenerative medicine [
      • Caplan A.I.
      Mesenchymal stem cells.
      ]. However, using allogeneic hMSCs from various sources for immunomodulatory purposes for acute inflammatory situations like treating severe acute GVHD, allogeneic cells may be an advantage and a prerequisite for storage until clinical use. In a mouse model, it was shown that allogeneic MSCs undergo perforin-dependent apoptosis by recipient cytotoxic T cells after infusion to reverse GVHD [
      • Galleu A.
      • et al.
      Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.
      ]. Furthermore, recipient phagocytes engulf apoptotic MSCs and produce IDO, which is necessary for T-cell suppression and to reverse GVHD [
      • Galipeau J.
      Macrophages at the nexus of mesenchymal stromal cell potency: The emerging role of chemokine cooperativity.
      ,
      • Kim J.
      • Hematti P.
      Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages.
      ,
      • Waterman R.S.
      • et al.
      A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.
      ,
      • Maggini J.
      • et al.
      Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.
      ].
      A study using Transwell experiments suggested that hDSCs in direct contact inhibit T-cell activation in MLC [
      • Erkers T.
      • et al.
      Decidual stromal cells promote regulatory T cells and suppress alloreactivity in a cell contact-dependent manner.
      ]. This diverged with hBM-MSCs where MLC was suppressed also by soluble factors, when separated by a Transwell membrane. In the present study, it seems that the hDSC inhibition in the MLC assay can mainly occur in direct contact and to a lesser degree by soluble mediators when separated by a Transwell membrane (Figure 3,A). However, to see this soluble mediated suppression the hDSCs need to be stimulated and have cell-to-cell contact with responder T cells. PD-L1 expressed by hDSCs reduces T-cell activation and proliferation through binding to the programmed death-1 receptors expressed on human T cells. It has been shown that this suppression was abolished when PD-L1 was blocked. This suggests a contact-dependent mechanism between programmed death-1 and PD-L1. Neutralization of interferon-γ, indoleamine 2,3-deoxigenase and prostaglandine-E2 resulted in elimination of immunosuppression of MLC by DSCs [
      • Erkers T.
      • et al.
      Decidual stromal cells promote regulatory T cells and suppress alloreactivity in a cell contact-dependent manner.
      ]. These blocking experiments suggest that both soluble factors and direct contact may be involved in DSCs immunosuppression in alloreactive in vitro assay. The latter is supported by the experiments when T cells, autologous to the responder T cell in the lower chamber, were added over the insert in vicinity of hDSCs in the Transwell MLC experiments. In this setting, T-cell proliferation in the lower chamber was significantly suppressed (Figure 3,A). This suggests that the crosstalk between hT cells and hDSCs results in soluble factor secretion, such as exosomes, inhibitory cytokines like IDO and interferon-γ, which suppress alloreactivity [
      • Erkers T.
      • et al.
      Decidual stromal cells promote regulatory T cells and suppress alloreactivity in a cell contact-dependent manner.
      ,
      • Meisel R.
      • et al.
      Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation.
      ].
      When autologous CD14+ monocyte cells were added back to the T cells stimulated with CD3/CD28 cocultured with DSCs as modulator cells, the immunosuppressive effects of hDSCs were decreased (Figure 3A,B). These findings may be because monocytes have some protective mechanism to prevent immune suppression via stromal cells. This finding needs to be explored in more detail. We speculate that in some cases with CD14+ cell populations were more polarized towards a M1 phenotype, which resulted in intervention of immunosuppression by hDSCs. In individuals in whom the CD14+ cell population was more polarized toward the M2 phenotype, the immunosuppressive effect of hDSCs in MLC was maintained. To elucidate this hypothesis, further experiments adding M1 or M2 phenotypes to these experiments are needed.
      Several studies have shown the pivotal role of monocytes/macrophages for hMSCs induced immunosuppression [
      • Galleu A.
      • et al.
      Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.
      ,
      • Cheung T.S.
      • Dazzi F.
      Mesenchymal-myeloid interaction in the regulation of immunity.
      ,
      • Chiossone L.
      • et al.
      Mesenchymal Stromal Cells Induce Peculiar Alternatively Activated Macrophages Capable of Dampening Both Innate and Adaptive Immune Responses.
      ,
      • Braza F.
      • et al.
      Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma.
      ,
      • Melief S.M.
      • et al.
      Multipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by production of interleukin-6.
      ]. Most of these studies are performed using hBM-MSCs. The present experiments demonstrate that monocytes also play an important role in the profound immunosuppression induced by hDSCs [
      • Rasmusson I.
      • et al.
      Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells.
      ].
      NK cells can both promote and prevent alloreactivity such as GVHD [
      • Simonetta F.
      • Alvarez M.
      • Negrin R.S.
      Natural Killer Cells in Graft-versus-Host-Disease after Allogeneic Hematopoietic Cell Transplantation.
      ]. NK cells have also a graft-versus-leukemia effect (GVL) [

      Barrett, A.J., Understanding and harnessing the graft-versus-leukaemia effect. 2008. 142(6): p. 877-888.

      ,
      • Lamb M.G.
      • et al.
      Natural killer cell therapy for hematologic malignancies: successes, challenges, and the future.
      ]. We previously reported that hBM-MSCs did not affect NK-cell–mediated lysis of K562 cells [
      • Rasmusson I.
      • et al.
      Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells.
      ]. We wanted to study whether hDSCs affect NK-cell proliferation induced by IL-2 and allogeneic PBL (Figure 3,C). In these situations, the addition of hDSCs increased NK-cell proliferation. The addition of autologous CD14+ monocyte cells did not affect this increased stimulation induced by hDSCs. It is possible that hDSCs express different ligands that are recognized by activating NK-cell receptors, which may trigger NK-cell–mediated alloreactivity. The findings may also suggest that treatment with hDSCs may not decrease the GVL effect induced by NK cells after allogeneic HCT. Additional studies may be required using in vivo models to further clarify these cell interactions.
      François et al. [
      • François M.
      • et al.
      Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing.
      ] showed that cryo-preserved BM-MSCs could not suppress T-cell proliferation in vitro and suggested that this was a possible explanation why some patients did not respond clinically to hBM-MSCs immunosuppression. Another study confirmed that freshly cultured hBM-MSCs had a stronger immunosuppressive effect than thawed–frozen cells [
      • Moll G.
      • et al.
      Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?.
      ]. Our study indicated that thawed cryo-preserved DSCs are at least as good as freshly cultured DSCs for inhibition of T-cell proliferation. The findings in this study may suggest that hDSCs are more robust to freezing and thawing as opposed to hBM-MSCs. Another possibility is that our freezing and thawing technique is superior to preserve hDSC function compared with that performed by the other investigators. In the MLC, fresh and frozen–thawed DSCs showed a similar inhibition of PBMC proliferation. However, when PBMCs were stimulated with PHA, frozen–thawed DSCs had a slightly stronger immunosuppressive effect compared with fresh cells. This may be explained by MSCs inhibiting T-cell proliferation stimulated by allo-antigens and mitogens by different mechanisms [
      • Rasmusson I.
      • et al.
      Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms.
      ]. Furthermore, there were no differences in immunomodulatory capacity, cell counts and cell viability between the fresh and the frozen–thawed hDSCs. This may further show an advantage for hDSCs to be used for clinical therapy as compared to hBM-MSCs. It was reported that freshly cultured hBM-MSCs kept viability better in room temperature than frozen–thawed cells, after storage in normal saline [
      • Pal R.
      • Hanwate M.
      • Totey S.M.
      Effect of holding time, temperature and different parenteral solutions on viability and functionality of adult bone marrow-derived mesenchymal stem cells before transplantation.
      ]. When we compared freshly cultured and frozen–thawed hDSCs kept in saline supplemented with 5% human serum albumin and in room temperature for 24 h, the viability of the cells was the same and there was no difference between the freshly cultured and the frozen–thawed cells. Cells of good viability >95% at start of thawing or after expansion (Fresh), kept a good viability for 24 hours. Cells of poor viability remained poorer but may be re-cultured, expanded and subsequently improved. BM-MSCs and umbilical cord-MSCs, supplemented with 5% albumin, showed a negative effect on viability of short-term storage in room temperature [
      • Lane T.A.
      • et al.
      Liquid storage of marrow stromal cells.
      ,
      • Chen Y.
      • et al.
      Effects of storage solutions on the viability of human umbilical cord mesenchymal stem cells for transplantation.
      ].

      Conclusions

      Human DSCs is a robust stromal cell with a strong immunomodulatory effect in clinical practice. To improve clinical efficacy, we must explore underlying mechanisms and crosstalk with other immune cells. We have found that DSCs have a lower capacity to decrease mouse MLC activation compared with autologous or mMHC identical and haploidentical mMSCs. Moreover, CD14+ monocyte cells reduce the immune modulation by DSCs. hDSCs do not affect NK-cell activation, which may preserve the NK cell induced GVL effect which is important after HCT. Fresh or freeze–thawed DSCs were equally immunosuppressive and had similar viability for 24 hours.

      Funding

      The study was supported by grants from the Swedish Cancer Foundation ( CAN2018, 419 ) and the Cancer Society in Stockholm (181263). OR was the recipient of a Distinguished Professor Award from the Karolinska Institutet.

      Declaration of Competing Interest

      The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

      Author Contributions

      Conception and design of the study: BS, MW, DS, AA, and OR. Acquisition of data: BS, MW, DS, and AA. Analysis and interpretation of data: BS, MW, DS, and AA. Drafting or revising the manuscript: BS and OR. All authors have approved the final article.

      Acknowledgments

      We would like to thank Gunilla Tillinger for excellent typing of this manuscript.

      References

        • Friedenstein A.J.
        • Chailakhjan R.K.
        • Lalykina K.S.
        The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells.
        Cell Tissue Kinet. 1970; 3: 393-403
        • Campagnoli C.
        • et al.
        Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow.
        Blood. 2001; 98: 2396-2402
        • Moll G.
        • et al.
        Intravascular Mesenchymal Stromal/Stem Cell Therapy Product Diversification: Time for New Clinical Guidelines.
        Trends Mol Med. 2019; 25: 149-163
        • Ringden O.
        • et al.
        Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease.
        Transplantation. 2006; 81: 1390-1397
        • Hashmi S.
        • et al.
        Survival after mesenchymal stromal cell therapy in steroid-refractory acute graft-versus-host disease: systematic review and meta-analysis.
        Lancet Haematol. 2016; 3: e45-e52
        • Zheng G.
        • et al.
        Treatment of acute respiratory distress syndrome with allogeneic adipose-derived mesenchymal stem cells: a randomized, placebo-controlled pilot study.
        Respir Res. 2014; 15: 39
        • Sadeghi B.
        • et al.
        Conquering the cytokine storm in COVID-19-induced ARDS using placenta-derived decidua stromal cells.
        J Cell Mol Med. 2021; 25: 10554-10564
        • Wang Y.
        • et al.
        Clinical application of mesenchymal stem cells in rheumatic diseases.
        Stem Cell Res Ther. 2021; 12: 567
        • Zhuang X.
        • et al.
        Mesenchymal Stem Cell-Based Therapy as a New Approach for the Treatment of Systemic Sclerosis.
        Clin Rev Allergy Immunol. 2022;
        • Ringden O.
        • et al.
        Mesenchymal Stromal Cells for Enhancing Hematopoietic Engraftment and Treatment of Graft-Versus-Host Disease, Hemorrhages and Acute Respiratory Distress Syndrome.
        Front Immunol. 2022; 13839844
        • Bartholomew A.
        • et al.
        Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.
        Exp Hematol. 2002; 30: 42-48
        • Karlsson H.
        • et al.
        Stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro.
        Clin Exp Immunol. 2012; 167: 543-555
        • Jones S.
        • et al.
        The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells.
        J Immunol. 2007; 179: 2824-2831
        • Mincheva-Nilsson L.
        Immune cells and molecules in pregnancy: friends or foes to the fetus?.
        Expert Rev Clin Immunol. 2006; 2: 457-470
        • Sadeghi B.
        • et al.
        Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo.
        Cytotherapy. 2015; 17: 1732-1745
        • Ringden O.
        • et al.
        Placenta-Derived Decidua Stromal Cells for Treatment of Severe Acute Graft-Versus-Host Disease.
        Stem Cells Transl Med. 2018; 7: 325-331
        • Ringden O.
        • et al.
        Fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease.
        Stem Cells. 2013; 31: 592-601
        • Erkers T.
        • et al.
        Decidual stromal cells promote regulatory T cells and suppress alloreactivity in a cell contact-dependent manner.
        Stem Cells Dev. 2013; 22: 2596-2605
        • Moll G.
        • et al.
        Different Procoagulant Activity of Therapeutic Mesenchymal Stromal Cells Derived from Bone Marrow and Placental Decidua.
        Stem Cells Dev. 2015; 24: 2269-2279
        • Kazemi S.
        • et al.
        Growth kinetic comparison of Human Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue and Decidua.
        Med Sci. 2020; 24: 223-234
        • Parekkadan B.
        • et al.
        Bone marrow stromal cell transplants prevent experimental enterocolitis and require host CD11b+ splenocytes.
        Gastroenterology. 2011; 140: 966-975
        • Galleu A.
        • et al.
        Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation.
        Sci Transl Med. 2017; 9
        • Hoogduijn M.J.
        • et al.
        Effects of Freeze-Thawing and Intravenous Infusion on Mesenchymal Stromal Cell Gene Expression.
        Stem Cells Dev. 2016; 25: 586-597
        • Chinnadurai R.
        • et al.
        Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.
        Stem Cells. 2016; 34: 2429-2442
        • Moll G.
        • et al.
        Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?.
        Stem Cells. 2014; 32: 2430-2442
        • Moll G.
        • Hoogduijn M.J.
        • Ankrum J.A.
        Editorial: Safety, Efficacy and Mechanisms of Action of Mesenchymal Stem Cell Therapies.
        Front Immunol. 2020; 11: 243
        • Mills C.D.
        • et al.
        M-1/M-2 macrophages and the Th1/Th2 paradigm.
        J Immunol. 2000; 164: 6166-6173
        • Koç O.N.
        • et al.
        Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.
        J Clin Oncol. 2000; 18: 307-316
        • Lazarus H.M.
        • et al.
        Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.
        Biol Blood Marrow Transplant. 2005; 11: 389-398
        • Le Blanc K.
        • et al.
        Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.
        Scand J Immunol. 2003; 57: 11-20
        • Le Blanc K.
        • et al.
        Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.
        Lancet. 2004; 363: 1439-1441
        • Le Blanc K.
        • et al.
        Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.
        Lancet. 2008; 371: 1579-1586
        • Martin P.J.
        • et al.
        Prochymal Improves Response Rates In Patients With Steroid-Refractory Acute Graft Versus Host Disease (SR-GVHD) Involving The Liver And Gut: Results Of A Randomized, Placebo-Controlled, Multicenter Phase III Trial In GVHD.
        Biol Blood Marrow Transplant. 2010; 16: S169-S170
        • Kebriaei P.
        • et al.
        A Phase 3 Randomized Study of Remestemcel-L versus Placebo Added to Second-Line Therapy in Patients with Steroid-Refractory Acute Graft-versus-Host Disease.
        Biol Blood Marrow Transplant. 2020; 26: 835-844
        • Aronsson-Kurttila W.
        • et al.
        Placenta-Derived Decidua Stromal Cells for Hemorrhagic Cystitis after Stem Cell Transplantation.
        Acta Haematol. 2018; 139: 106-114
        • Kaipe H.
        • et al.
        Immunogenicity of decidual stromal cells in an epidermolysis bullosa patient and in allogeneic hematopoietic stem cell transplantation patients.
        Stem Cells Dev. 2015; 24: 1471-1482
        • Watanabe H.
        • et al.
        Innate immune response in Th1- and Th2-dominant mouse strains.
        Shock. 2004; 22: 460-466
        • Caplan A.I.
        Mesenchymal stem cells.
        J Orthop Res. 1991; 9: 641-650
        • Galipeau J.
        Macrophages at the nexus of mesenchymal stromal cell potency: The emerging role of chemokine cooperativity.
        Stem Cells. 2021; 39: 1145-1154
        • Kim J.
        • Hematti P.
        Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages.
        Exp Hematol. 2009; 37: 1445-1453
        • Waterman R.S.
        • et al.
        A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype.
        PLoS One. 2010; 5: e10088
        • Maggini J.
        • et al.
        Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.
        PLoS One. 2010; 5: e9252
        • Meisel R.
        • et al.
        Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation.
        Blood. 2004; 103: 4619-4621
        • Cheung T.S.
        • Dazzi F.
        Mesenchymal-myeloid interaction in the regulation of immunity.
        Semin Immunol. 2018; 35: 59-68
        • Chiossone L.
        • et al.
        Mesenchymal Stromal Cells Induce Peculiar Alternatively Activated Macrophages Capable of Dampening Both Innate and Adaptive Immune Responses.
        Stem Cells. 2016; 34: 1909-1921
        • Braza F.
        • et al.
        Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma.
        Stem Cells,. 2016; 34: 1836-1845
        • Melief S.M.
        • et al.
        Multipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by production of interleukin-6.
        Haematologica. 2013; 98: 888-895
        • Rasmusson I.
        • et al.
        Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells.
        Transplantation. 2003; 76: 1208-1213
        • Simonetta F.
        • Alvarez M.
        • Negrin R.S.
        Natural Killer Cells in Graft-versus-Host-Disease after Allogeneic Hematopoietic Cell Transplantation.
        Front Immunol. 2017; 8: 465
      1. Barrett, A.J., Understanding and harnessing the graft-versus-leukaemia effect. 2008. 142(6): p. 877-888.

        • Lamb M.G.
        • et al.
        Natural killer cell therapy for hematologic malignancies: successes, challenges, and the future.
        Stem Cell Res Ther. 2021; 12: 211
        • François M.
        • et al.
        Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing.
        Cytotherapy. 2012; 14: 147-152
        • Rasmusson I.
        • et al.
        Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms.
        Exp Cell Res. 2005; 305: 33-41
        • Pal R.
        • Hanwate M.
        • Totey S.M.
        Effect of holding time, temperature and different parenteral solutions on viability and functionality of adult bone marrow-derived mesenchymal stem cells before transplantation.
        J Tissue Eng Regen Med. 2008; 2: 436-444
        • Lane T.A.
        • et al.
        Liquid storage of marrow stromal cells.
        Transfusion. 2009; 49: 1471-1481
        • Chen Y.
        • et al.
        Effects of storage solutions on the viability of human umbilical cord mesenchymal stem cells for transplantation.
        Cell Transplant. 2013; 22: 1075-1086