LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration.

Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML.

The use of therapeutic plasma exchange in systemic loxoscelism induced treatment resistant hemolytic anemia: A case report.

Brown recluse spider bites can lead to severe reactions such as skin necrosis,hemolytic anemia, and multiorgan failure, which can be life-threatening. Therapeutic plasma exchange has been reported to provide clinical benefit for such cases. In thisreport, we present a case of a brown recluse spider bite that was successfully treated with therapeutic plasma exchange and compare it with previous case reports.

Hemoglobin S target of <50% as compared to 30% in chronic red cell exchange for secondary stroke prevention in sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) patients with a history of stroke are encouraged to receive chronic red blood cell exchange (RBCx) for stroke prevention. The American Society of Hematology guideline published in 2020 recommends an HbS target of <30%. However, this approach necessitates more frequent RBCx and more RBC units. UT Southwestern has devised a chronic exchange protocol that elevates the HbS target to <50% in patients with a low risk of stroke. STUDY DESIGN: This retrospective chart review study reviewed the medical records of patients receiving chronic RBCx with a target of HbS <50% over the past 10-year period to assess the safety of maintaining higher HbS targets in SCD patients with a low risk of cerebrovascular accidents (CVA). RESULTS: Among 49 SCD patients in the chronic RBCx program for secondary stroke prevention, 33 patients were maintained on an HbS target of <50% (average measured: 35.4%) for the duration of RBCx program enrollment (median 93.0 months, 95% CI, 83-99). Stroke or transient ischemic attack (TIA) clearly attributable to changing target HbS had not been identified among the 33 study subjects. Seven patients experienced conversion between the HbS targets of <50% and <30% HbS target. Significant reductions were observed in the frequency of RBCx and usage of blood volume in four of them. CONCLUSION: The findings suggest that liberalizing the HbS target could confer clinical flexibility without increasing the risk of CVA in a selective population. Further studies to fully evaluate the potential benefits of this approach are indicated.

The role of an augmentative plating in the management of femoral subtrochanteric nonunion.

INTRODUCTION: Owing to its distinct biomechanical properties, nonunion is common (7-20%) after intramedullary (IM) nailing of subtrochanteric femoral fractures. Unlike diaphyseal nonunion, it is difficult to provide sufficient stability by exchanging nailing alone in subtrochanteric nonunion. This study investigated the clinical outcomes of femoral subtrochanteric nonunion initially treated with an IM nail and subsequently managed with minimally invasive augmentative plate fixation. MATERIALS AND METHODS: Nineteen patients were enrolled retrospectively. The mechanisms of initial injury were traffic accidents in 8, falls from a height in seven, and slipping in two patients. Two patients with atypical subtrochanteric femoral fractures without a specific trauma history were further included. All patients underwent IM nailing as the index operation. Nonunion surgery was performed an average of 45.2 weeks after the initial surgery. In cases of hardware damage and/or atrophic nonunion, exchange nailing and bone grafting were performed in addition to augmentative plating, as necessary. Conversely, augmentative plating alone was performed in cases of hypertrophic nonunion without any failure of the preexisting IM nail or malalignment. A narrow locking compression plate was fixed after contouring according to the shape of the proximal femur. The mean follow-up period was 36.1 months. RESULTS: Bony union was achieved in 18/19 patients (94.7%), at an average of 19.8 weeks after nonunion surgery. In the case that did not heal even after exchange nailing, additional plating and bone grafting, further autogenous bone grafting was required after 11 months, which healed uneventfully. There were 2 cases of soft tissue irritation over the plate, but no major complications were observed. CONCLUSIONS: Additional plate augmentation over a retained IM nail yields satisfactory outcomes in terms of the bony union in subtrochanteric nonunion. Given its expected biomechanical superiority and relatively easy surgical technique, it may be a reasonable option for the management of femoral subtrochanteric nonunion.

Mathematical calculation of lifespan of transfused RBCs in sickle cell disease patients.

BACKGROUND: Transfusion of donor red blood cells (RBCs) remains an important part of management of sickle cell disease (SCD). However, the survival characteristics of transfused donor RBCs in SCD patients have not been well studied. We sought to calculate survival kinetics of transfused RBCs in SCD patients since it is unclear whether transfused RBCs get destroyed at faster rate as innocent bystander or persist longer due to decreased destruction capacity such as functional splenectomy. STUDY DESIGN: and methods Forty-one SCD patients who had undergone at least 3 RBC exchange procedures were inlcuded. Interval between the procedures, both pre-procedure and post procedure hematocrits, HbA% and HbS% were collected. We developed a mathematical model to calculate RBC lifespan for donor RBCs. RESULTS: Donor RBCs exhibited average lifespan of about 120days (121.1±13.9 days), which was similar to reported survival of RBCs in normal recipients. However, significant variation between patients were observed with lifespan ranging from 75.6-148.5 days. Intrapersonal variations were small in most cases. CONCLUSION: The calculated survival of donor RBCs in SCD recipient, based on certain laboratory values, appears to be similar to that of normal recipient. However, inter-personal variations were large, suggesting different RBC kinetics in a subset of patients, which calls for further research to better understand underlying pathophysiology. This knowledge of RBC survival would be very helpful in individualized management of patients on chronic RBCx.

Single-molecule analysis reveals widespread structural variation in multiple myeloma.

Multiple myeloma (MM), a malignancy of plasma cells, is characterized by widespread genomic heterogeneity and, consequently, differences in disease progression and drug response. Although recent large-scale sequencing studies have greatly improved our understanding of MM genomes, our knowledge about genomic structural variation in MM is attenuated due to the limitations of commonly used sequencing approaches. In this study, we present the application of optical mapping, a single-molecule, whole-genome analysis system, to discover new structural variants in a primary MM genome. Through our analysis, we have identified and characterized widespread structural variation in this tumor genome. Additionally, we describe our efforts toward comprehensive characterization of genome structure and variation by integrating our findings from optical mapping with those from DNA sequencing-based genomic analysis. Finally, by studying this MM genome at two time points during tumor progression, we have demonstrated an increase in mutational burden with tumor progression at all length scales of variation.

Human Mesenchymal Stem Cell-Educated Macrophages Are a Distinct High IL-6-Producing Subset that Confer Protection in Graft-versus-Host-Disease and Radiation Injury Models.

Mesenchymal stem cells (MSCs) have immunosuppressive and tissue repair properties, but clinical trials using MSCs to prevent or treat graft-versus-host disease (GVHD) have shown mixed results. Macrophages (MØs) are important regulators of immunity and can promote tissue regeneration and remodeling. We have previously shown that MSCs can educate MØs toward a unique anti-inflammatory immunophenotype (MSC-educated MØs [MEMs]); however, their implications for in vivo models of inflammation have not been studied yet. We now show that in comparison with MØs, MEMs have increased expression of the inhibitory molecules PD-L1, PD-L2, in addition to markers of alternatively activated MØs: CD206 and CD163. RNA-Seq analysis of MEMs, as compared with MØs, show a distinct gene expression profile that positively correlates with multiple pathways important in tissue repair. MEMs also show increased expression of IL-6, transforming growth factor-ß, arginase-1, CD73, and decreased expression of IL-12 and tumor necrosis factor-α. We show that IL-6 secretion is controlled in part by the cyclo-oxygenase-2, arginase, and JAK1/STAT1 pathway. When tested in vivo, we show that human MEMs significantly enhance survival from lethal GVHD and improve survival of mice from radiation injury. We show these effects could be mediated in part through suppression of human T cell proliferation and may have attenuated host tissue injury in part by enhancing murine fibroblast proliferation. MEMs are a unique MØ subset with therapeutic potential for the management of GVHD and/or protection from radiation-induced injury.

TPL2 kinase regulates the inflammatory milieu of the myeloma niche.

Targeted modulation of microenvironmental regulatory pathways may be essential to control myeloma and other genetically/clonally heterogeneous cancers. Here we report that human myeloma-associated monocytes/macrophages (MAM), but not myeloma plasma cells, constitute the predominant source of interleukin-1ß (IL-1ß), IL-10, and tumor necrosis factor-α at diagnosis, whereas IL-6 originates from stromal cells and macrophages. To dissect MAM activation/cytokine pathways, we analyzed Toll-like receptor (TLR) expression in human myeloma CD14(+) cells. We observed coregulation of TLR2 and TLR6 expression correlating with local processing of versican, a proteoglycan TLR2/6 agonist linked to carcinoma progression. Versican has not been mechanistically implicated in myeloma pathogenesis. We hypothesized that the most readily accessible target in the versican-TLR2/6 pathway would be the mitogen-activated protein 3 (MAP3) kinase, TPL2 (Cot/MAP3K8). Ablation of Tpl2 in the genetically engineered in vivo myeloma model, Vκ*MYC, led to prolonged disease latency associated with plasma cell growth defect. Tpl2 loss abrogated the "inflammatory switch" in MAM within nascent myeloma lesions and licensed macrophage repolarization in established tumors. MYC activation/expression in plasma cells was independent of Tpl2 activity. Pharmacologic TPL2 inhibition in human monocytes led to dose-dependent attenuation of IL-1ß induction/secretion in response to TLR2 stimulation. Our results highlight a TLR2/6-dependent TPL2 pathway as novel therapeutic target acting nonautonomously through macrophages to control myeloma progression.

Cardiopulmonary and histological characterization of an acute rat lung injury model demonstrating safety of mesenchymal stromal cell infusion.

BACKGROUND AIMS: In the field of cellular therapy, potential cell entrapment in the lungs following intravenous administration in a compromised or injured pulmonary system is an important concern that requires further investigation. We developed a rat model of inflammatory and fibrotic lung disease to mimic the human clinical condition of obliterative bronchiolitis (OB) and evaluate the safety of intravenous infusion of mesenchymal stromal cells (MSCs). This model was used to obtain appropriate safety information and functional characterization to support the translation of an ex vivo-generated cellular product into human clinical trials. To overcome spontaneous recovery and size limitations associated with current animal models, we used a novel multiple dose bleomycin strategy to induce lasting lung injury in rats. METHODS: Intratracheal instillation of bleomycin was administered to rats on multiple days. MSCs were intravenously infused 7 days apart. Detailed pulmonary function tests including forced expiratory volume, total lung capacity, and invasive hemodynamic measurements were conducted to define the representative disease model and monitor cardiopulmonary hemodynamic consequences of the cell infusion. Post-euthanasia assessments included a thorough evaluation of lung morphology and histopathology. RESULTS: The double dose bleomycin instillation regimen resulted in severe and irreversible lung injury and fibrosis. Cardiopulmonary physiological monitoring reveled that no adverse events could be attributed to the cell infusion process. DISCUSSION: Although our study did not show the infusion of MSCs to result in an improvement in lung function or rescue of damaged tissue this study does confirm the safety of MSC infusion into damaged lungs.

Fibroblasts and Mesenchymal Stromal/Stem Cells Are Phenotypically Indistinguishable.

BACKGROUND/AIMS: Human mesenchymal stromal/stem cells (MSCs), derived from many different tissues, are characterized by a fibroblast-like morphology, the expression of certain cell surface markers and their ability to differentiate into adipocytes, chondrocytes and osteoblasts. A number of studies have shown that MSCs share many characteristics with fibroblasts; however, there is no well-defined set of phenotypic characteristics that could distinguish between these 2 types of cells. METHODS: We used 4 well-established human fibroblast strains from 3 different tissue sources and several human MSC strains from 2 different tissue sources to compare the phenotypic and immunological characteristics of these cells. RESULTS: Fibroblast strains had a similar morphology to MSCs, expressed the same cell surface markers as MSCs and could also differentiate into adipocytes, chondrocytes and osteoblasts. Also, similar to MSCs, these fibroblasts were capable of suppressing T cell proliferation and modulating the immunophenotype of macrophages. We also show that MSCs deposit extracellular matrices of collagen type I and fibronectin, and express FSP1 in patterns similar to fibroblasts. CONCLUSIONS: Based on currently accepted definitions for cultured human MSCs and fibroblasts, we could not find any immunophenotypic property that could make a characteristic distinction between MSCs and fibroblasts.

Comparison of Spectra Optia and COBE Spectra apheresis systems' performances for red blood cell exchange procedures.

BACKGROUND: Spectra Optia (Terumo BCT, Lakewood, CO) was FDA approved for red blood cell exchange (RBCx) procedures in January 2014 and is expected to replace COBE spectra (Terumo BCT) very soon in the USA. The performance characteristics of these devices for Isovolemic Hemodilution (IHD-RBCx) procedure were compared in this study. METHODS: A total of 114 IHD-RBCx procedures from 19 patients were analyzed. For every patient, three procedures on each device with similar pre-procedure hematocrits were compared. Pre and post procedure laboratory parameters compared were hemoglobin S (HbS), hematocrits (Hct), platelet counts and fraction of cells remaining (FCR). Statistical analysis was performed using t-test adjusted by the Holm-Bonferroni method to reduce family-wise error rate. RESULTS: There were no significant differences between these two devices in regards to HbS, Hct, FCR and platelet counts (p = > 0.05). However, rinseback volume (124.2 ± 8.9 ml) and normal saline replacement volume during IHD phase (296.1 ± 97.2 ml) were lower in Spectra Optia as compared to COBE Spectra (337 ± 33.8 ml and 326.6 ± 105.2 ml, p value <0.001 and 0.030 respectively). Spectra Optia had a longer run time (107.1 ± 15.9 min vs 123.8 ± 19.6 min, p value <0.001) overall. CONCLUSIONS: Performance characteristics of Spectra Optia for HbS, Hct and FCR were similar to COBE Spectra for IHD-RBCx. IHD-RBCx procedure on Optia required less normal saline replacement volume and rinse back volume but with overall longer procedure run time.

Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model.

BACKGROUND AIMS: Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. METHODS: We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4(+) fraction of CD34(+) HSCs, we could improve engraftment. Human cord blood-derived CD34(+) cells and human bone marrow-derived MSCs were used for these studies. RESULTS: When MSCs were transplanted 1 week before CD34(+) cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34(+) cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4(+) cells in the CD34(+) population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. CONCLUSIONS: Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4-SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.

Galectin 7 leads to a relative reduction in CD4+ T cells, mediated by PD-1.

The role of glycan-binding proteins as an activator of immune regulatory receptors has gained attention recently. We report that galectin 7 reduced CD4+ T cell percentage in both in vitro culture and mouse tumor models. Immunohistochemical staining of esophageal cancer patient samples showed a lower percentage of CD4+ cells in the galectin 7 high area. The lack of CD4+ T cell depletion by galectin 7 in PD-1 knockout mice supports the role of PD-1 in mediating the effects of galectin 7. The binding assays demonstrate that galectin 7 binds to the N-glycosylation of PD-1 on N74 and N116 sites and leads to the recruitment of SHP-2. NFAT suppressive activity of galectin 7 was abrogated upon overexpression of the dominant negative SHP-2 mutant or inhibition of PD-1 by siRNA. Glycosylation of PD-1 has been reported to play a critical role in surface expression, stability, and interaction with its ligand PD-L1. This report further expands the significance of PD-1 glycosylation and suggests that galectin 7, a glycan-binding protein, interacts with the immune regulatory receptor PD-1 through glycosylation recognition.

LILRB3 Supports Immunosuppressive Activity of Myeloid Cells and Tumor Development.

The existing T cell-centered immune checkpoint blockade therapies have been successful in treating some but not all patients with cancer. Immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSC), that inhibit antitumor immunity and support multiple steps of tumor development are recognized as one of the major obstacles in cancer treatment. Leukocyte Ig-like receptor subfamily B3 (LILRB3), an immune inhibitory receptor containing tyrosine-based inhibitory motifs (ITIM), is expressed solely on myeloid cells. However, it is unknown whether LILRB3 is a critical checkpoint receptor in regulating the activity of immunosuppressive myeloid cells, and whether LILRB3 signaling can be blocked to activate the immune system to treat solid tumors. Here, we report that galectin-4 and galectin-7 induce activation of LILRB3 and that LILRB3 is functionally expressed on immunosuppressive myeloid cells. In some samples from patients with solid cancers, blockade of LILRB3 signaling by an antagonistic antibody inhibited the activity of immunosuppressive myeloid cells. Anti-LILRB3 also impeded tumor development in myeloid-specific LILRB3 transgenic mice through a T cell-dependent manner. LILRB3 blockade may prove to be a novel approach for immunotherapy of solid cancers.

MAP3K8 kinase regulates myeloma growth by cell-autonomous and non-autonomous mechanisms involving myeloma-associated monocytes/macrophages.

Benefit from cytotoxic therapy in myeloma may be limited by the persistence of residual tumour cells within protective niches. We have previously shown that monocytes/macrophages acquire a proinflammatory transcriptional profile in the myeloma microenvironment. Here we report constitutive activation of MAP3K8 kinase-dependent pathways that regulate the magnitude and extent of inflammatory activity of monocytes/macrophages within myeloma niches. In myeloma tumour cells, MAP3K8 acts as mitogen-induced MAP3K in mitosis and is required for TNFα-mediated ERK activation. Pharmacological MAP3K8 inhibition results in dose-dependent, tumour cell-autonomous apoptosis despite contact with primary stroma. MAP3K8 blockade may disrupt crucial macrophage-tumour cell interactions within myeloma niches.

Comparison of breast and abdominal adipose tissue mesenchymal stromal/stem cells in support of proliferation of breast cancer cells.

In this study, we compared MSCs from breast and abdominal tissue in terms of their expression of genes deemed important in the support of breast cancer growth and their effect on gene profile of macrophages after coculture. In addition, we investigated the role of MSCs, alone or in combination with macrophages, on proliferation of breast cancer cell lines. Our results show that MSCs derived from breast and abdominal adipose tissues have a comparable gene expression profile, have similar effect on gene expression of macrophages, and are comparable in supporting breast cancer cell line proliferation.

Comparative analysis of adipose-derived mesenchymal stem cells isolated from abdominal and breast tissue.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSC) may have a potential dual role in soft tissue augmentation by suppressing inflammation and promoting regeneration. Due to these properties, there is increasing interest in their potential use in autologous fat grafting, particularly to the breast. OBJECTIVES: The authors isolate and compare ADSC derived from abdominal and breast tissues with a hypothesis that different adipose tissue sources may demonstrate different functional characteristics affecting outcomes in autologous cell transplantation in reconstructive and aesthetic surgery. METHODS: Adipose-derived mesenchymal stem cells from abdominal and breast tissues were isolated and compared in terms of surface marker expression, differentiation capabilities, and both fibroblast growth factor (FGF) and receptor expression. Immunophenotype of macrophages was also investigated using cell surface markers following a 7-day co-culture period with ADSC. RESULTS: Results showed similar cell surface phenotype and multilineage differentiation capabilities of ADSC derived from abdominal and breast tissues. Variations of FGF expression were demonstrated on reverse transcription polymerase chain reaction, with a significantly higher expression of FGF2 seen in breast ADSC. Following the 7-day co-culture period, increased expression of the anti-inflammatory surface marker CD206 was identified, with decreased CD16 and human leukocyte antigen-DR on macrophages co-cultured with ADSC compared with controls. CONCLUSIONS: The data indicate similarities between ADSC derived from abdominal and breast tissues. Significant differences were seen, however, in the expression of FGF2, which is important in angiogenesis and wound healing. The results support the utility of ADSC in cell-based therapies such as autologous fat grafting.

Macrophages and mesenchymal stromal cells support survival and proliferation of multiple myeloma cells.

Multiple myeloma (MM) is characterized by almost exclusive tropism of malignant cells for the bone marrow (BM) milieu. The survival and proliferation of malignant plasma cells have been shown to rely on interactions with nonmalignant stromal cells, in particular mesenchymal stromal cells (MSCs), in the BM microenvironment. However, the BM microenvironment is composed of a diverse array of cell types. This study examined the role of macrophages, an abundant component of BM stroma, as a potential niche component that supports malignant plasma cells. We investigated the proliferation of MM tumour cell lines when cultured alone or together with MSCs, macrophages, or a combination of MSCs and macrophages, using the carboxyfluorescein succinimidyl ester assay. Consistently, we observed increased proliferation of MM cell lines in the presence of either MSCs or macrophages compared to cell line-only control. Furthermore, the combined co-culture of MSCs plus macrophages induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, MSCs and macrophages decreased the rate of apoptosis of myeloma cells. Our in vitro studies provide evidence that highlights the role of macrophages as a key component of the BM microenvironment facilitating the growth of malignant plasma cells in MM.

Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. METHODS: We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. RESULTS: Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts.

Mesenchymal stromal cells are present in the heart and promote growth of adult stem cells in vitro.

BACKGROUND AIMS: For many years the human heart has been considered a terminally differentiated organ with no regenerative potential after injury. Recent studies, however, have cast doubt on this long-standing dogma. The objective of this study was to investigate the presence of and characterize mesenchymal stromal cells (MSC) in the adult mouse heart. The impact of MSC on growth and differentiation of adult cardiac stem cells (CSC) was also analyzed. METHODS: A combination of lineage-negative/c-kit-negative (Lin(-)/c-kit(-)) immunoselection with a plastic-adhesion technique was used to isolate cardiac-derived MSC. The differentiation capacity and expression of surface markers were analyzed. To investigate the impact of MSC on growth and differentiation of adult CSC, Green Fluorescent Protein (GFP(+)) adult CSC were co-cultured with GFP(-) cardiac-derived MSC. RESULTS: MSC were present in the adult mouse heart and they met the criteria established to define mouse MSC. They expressed surface markers and were able to differentiate, in a controlled manner, into multiple lineages. In addition, cardiac-derived MSC promoted the survival and expansion of adult CSC in vitro. CONCLUSIONS: MSC can be isolated from the mouse heart and they promote growth and differentiation of adult CSC. The findings from this study could have a significant beneficial impact on future heart failure treatment. Co-culture and co-implantation of cardiac-derived MSC with adult CSC could provide extensive cardiac regeneration and maintenance of the CSC population after implanted into the heart.