A unique immune signature in blood separates therapy-refractory from therapy-responsive acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is an immune cell‒driven, potentially lethal complication of allogeneic hematopoietic stem cell transplantation affecting diverse organs, including the skin, liver, and gastrointestinal (GI) tract. We applied mass cytometry (CyTOF) to dissect circulating myeloid and lymphoid cells in children with severe (grade III-IV) aGVHD treated with immune suppressive drugs alone (first-line therapy) or in combination with mesenchymal stromal cells (MSCs; second-line therapy). These results were compared with CyTOF data generated in children who underwent transplantation with no aGVHD or age-matched healthy control participants. Onset of aGVHD was associated with the appearance of CD11b+CD163+ myeloid cells in the blood and accumulation in the skin and GI tract. Distinct T-cell populations, including TCRγδ+ cells, expressing activation markers and chemokine receptors guiding homing to the skin and GI tract were found in the same blood samples. CXCR3+ T cells released inflammation-promoting factors after overnight stimulation. These results indicate that lymphoid and myeloid compartments are triggered at aGVHD onset. Immunoglobulin M (IgM) presumably class switched, plasmablasts, and 2 distinct CD11b- dendritic cell subsets were other prominent immune populations found early during the course of aGVHD in patients refractory to both first- and second-line (MSC-based) therapy. In these nonresponding patients, effector and regulatory T cells with skin- or gut-homing receptors also remained proportionally high over time, whereas their frequencies declined in therapy responders. Our results underscore the additive value of high-dimensional immune cell profiling for clinical response evaluation, which may assist timely decision-making in the management of severe aGVHD.

A robust and standardized method to isolate and expand mesenchymal stromal cells from human umbilical cord.

BACKGROUND AIMS: Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, we report on a robust and standardized method for hUC-MSC isolation and expansion. METHODS: Using 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Furthermore, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics. RESULTS: We demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of ∼98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity. CONCLUSIONS: We present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies.

Autologous bone marrow-derived mesenchymal stromal cell therapy with early tacrolimus withdrawal: The randomized prospective, single-center, open-label TRITON study.

After renal transplantation, there is a need for immunosuppressive regimens which effectively prevent allograft rejection, while preserving renal function and minimizing side effects. From this perspective, mesenchymal stromal cell (MSC) therapy is of interest. In this randomized prospective, single-center, open-label trial, we compared MSCs infused 6 and 7 weeks after renal transplantation and early tacrolimus withdrawal with a control tacrolimus group. Primary end point was quantitative evaluation of interstitial fibrosis in protocol biopsies at 4 and 24 weeks posttransplant. Secondary end points included acute rejection, graft loss, death, renal function, adverse events, and immunological responses. Seventy patients were randomly assigned of which 57 patients were included in the final analysis (29 MSC; 28 controls). Quantitative progression of fibrosis failed to show benefit in the MSC group and GFR remained stable in both groups. One acute rejection was documented (MSC group), while subclinical rejection in week 24 protocol biopsies occurred in seven patients (four MSC; three controls). In the MSC group, regulatory T cell numbers were significantly higher compared to controls (p = .014, week 24). In conclusion, early tacrolimus withdrawal with MSC therapy was safe and feasible without increased rejection and with preserved renal function. MSC therapy is a potentially useful approach after renal transplantation.

Cartilage from human-induced pluripotent stem cells: comparison with neo-cartilage from chondrocytes and bone marrow mesenchymal stromal cells.

Cartilage has little intrinsic capacity for repair, so transplantation of exogenous cartilage cells is considered a realistic option for cartilage regeneration. We explored whether human-induced pluripotent stem cells (hiPSCs) could represent such unlimited cell sources for neo-cartilage comparable to human primary articular chondrocytes (hPACs) or human bone marrow-derived mesenchymal stromal cells (hBMSCs). For this, chondroprogenitor cells (hiCPCs) and hiPSC-derived mesenchymal stromal cells (hiMSCs) were generated from two independent hiPSC lines and characterized by morphology, flow cytometry, and differentiation potential. Chondrogenesis was compared to hBMSCs and hPACs by histology, immunohistochemistry, and RT-qPCR, while similarities were estimated based on Pearson correlations using a panel of 20 relevant genes. Our data show successful differentiations of hiPSC into hiMSCs and hiCPCs. Characteristic hBMSC markers were shared between hBMSCs and hiMSCs, with the exception of CD146 and CD45. However, neo-cartilage generated from hiMSCs showed low resemblances when compared to hBMSCs (53%) and hPACs (39%) characterized by lower collagen type 2 and higher collagen type 1 expression. Contrarily, hiCPC neo-cartilage generated neo-cartilage more similar to hPACs (65%), with stronger expression of matrix deposition markers. Our study shows that taking a stepwise approach to generate neo-cartilage from hiPSCs via chondroprogenitor cells results in strong similarities to neo-cartilage of hPACs within 3 weeks following chondrogenesis, making them a potential candidate for regenerative therapies. Contrarily, neo-cartilage deposited by hiMSCs seems more prone to hypertrophic characteristics compared to hPACs. We therefore compared chondrocytes derived from hiMSCs and hiCPCs with hPACs and hBMSCs to outline similarities and differences between their neo-cartilage and establish their potential suitability for regenerative medicine and disease modelling.

Human leukocyte antigen selected allogeneic mesenchymal stromal cell therapy in renal transplantation: The Neptune study, a phase I single-center study.

Mesenchymal stromal cells (MSC) hold promise as a novel immune-modulatory therapy in organ transplantation. First clinical studies have used autologous MSCs; however, the use of allogeneic "off-the-shelf" MSCs is more sustainable for broad clinical implementation, although with the risk of causing sensitization. We investigated safety and feasibility of allogeneic MSCs in renal transplantation, using a matching strategy that prevented repeated mismatches. Ten patients received two doses of 1.5 × 106 /kg allogeneic MSCs 6 months after transplantation in a single-center nonrandomized phase Ib trial, followed by lowering of tacrolimus (trough level 3 ng/mL) in combination with everolimus and prednisone. Primary end point was safety, measured by biopsy proven acute rejection (BPAR) and graft loss 12 months after transplantation. Immune monitoring was performed before and after infusion. No BPAR or graft loss occurred and renal function remained stable. One patient retrospectively had DSAs against MSCs, formed before infusion. No major alterations in T and B cell populations or plasma cytokines were observed upon MSC infusion. Administration of HLA selected allogeneic MSCs combined with low-dose tacrolimus 6 months after transplantation is safe at least in the first year after renal transplantation. This sets the stage to further explore the efficacy of third-party MSCs in renal transplantation.

What does cell therapy manufacturing cost? A framework and methodology to facilitate academic and other small-scale cell therapy manufacturing costings.

BACKGROUND AIMS: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies. METHODS: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€). RESULTS: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices. CONCLUSIONS: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs.

Preparing for cell culture scale-out: establishing parity of bioreactor- and flask-expanded mesenchymal stromal cell cultures.

BACKGROUND: Cell-based therapies have the potential to become treatment options for many diseases, but efficient scale-out of these therapies has proven to be a major hurdle. Bioreactors can be used to overcome this hurdle, but changing the culture method can introduce unwanted changes to the cell product. Therefore, it is important to establish parity between products generated using traditional methods versus those generated using a bioreactor. METHODS: Mesenchymal stromal cells (MSCs) are cultured in parallel using either traditional culture flasks, spinner vessels or a new bioreactor system. To investigate parity between the cells obtained from different methods, harvested cells are compared in terms of yield, phenotype and functionality. RESULTS: Bioreactor-based expansion yielded high cell numbers (222-510 million cells). Highest cell expansion was observed upon culture in flasks [average 5.0 population doublings (PDL)], followed by bioreactor (4.0 PDL) and spinner flasks (3.3 PDL). Flow cytometry confirmed MSC identity (CD73+, CD90+ and CD105+) and lack of contaminating hematopoietic cell populations. Cultured MSCs did not display genetic aberrations and no difference in differentiation and immunomodulatory capacity was observed between culture conditions. The response to IFNγ stimulation was similar for cells obtained from all culture conditions, as was the capacity to inhibit T cell proliferation. CONCLUSIONS: The new bioreactor technology can be used to culture large amounts of cells with characteristics equivalent to those cultured using traditional, flask based, methods.

TwinLIFE: The Twin Longitudinal Investigation of FEtal Discordance.

Lifelong health is thought to be partially set during intrauterine life by persistent epigenetic changes induced by the prenatal environment. To evaluate this hypothesis, we initiated a prospective longitudinal study in monochorionic (MC) twins: the TwinLIFE study. MC twins are monozygotic, thus in origin genetically identical, and share a single placenta. Although MC twins have many environmental factors in common, in one-third of the MC twin pairs, one fetus has significantly less access to nutrients and resources during pregnancy than its co-twin often resulting in a significant discordance in prenatal growth. Hence, MC twins constitute a unique natural experiment to study the influence of the prenatal environment on health. In TwinLIFE, we will chart intrapair differences in DNA methylation focusing on mesenchymal stromal cells isolated from cord as an advanced proxy of epigenetic dysregulation relevant for long-term health consequences. Next, we will follow up the MC twins for growth, cardiovascular and neurodevelopmental outcomes during childhood and evaluate the impact of an epigenetic signature at birth on future health. The current target is to include 100 MC twin pairs, but we aim to continue enrollment after procuring additional funding. TwinLIFE will not only address an unmet clinical need in the high-risk group of MC twins, but may also advance early-life strategies to prevent adverse growth, cardiovascular and neurodevelopmental outcomes in the general population.

HLA reduces killer cell Ig-like receptor expression level and frequency in a humanized mouse model.

NK cells use NK cell receptors to be able to recognize and eliminate infected, transformed, and allogeneic cells. Human NK cells are prevented from killing autologous healthy cells by virtue of inhibitory NKRs, primarily killer cell Ig-like receptors (KIR) that bind "self" HLA class I molecules. Individual NK cells stably express a selected set of KIR, but it is currently disputed whether the fraction of NK cells expressing a particular inhibitory KIR is influenced by the presence of the corresponding HLA ligand. The extreme polymorphism of the KIR and HLA loci, with wide-ranging affinities for individual KIR and HLA allele combinations, has made this issue particularly hard to tackle. In this study, we used a transgenic mouse model to investigate the effect of HLA on KIR repertoire and function in the absence of genetic variation inside and outside the KIR locus. These H-2K(b-/-) and H-2D(b-/-) mice lacked ligands for inhibitory Ly49 receptors and were transgenic for HLA-Cw3 and a KIR B haplotype. In this reductionist system, the presence of HLA-Cw3 reduced the frequency of KIR2DL2(+) cells, as well as the surface expression levels of KIR2DL2. In addition, in the presence of HLA-Cw3, the frequency of NKG2A(+) cells and the surface expression levels of NKG2A were reduced. In line with these findings, both transgene-encoded KIR and endogenous NKG2A contributed to the rejection of cells lacking HLA-Cw3. These findings support the idea that HLA influences the human KIR repertoire.

Twisting the theory on the origin of human umbilical cord coiling featuring monozygotic twins.

The human umbilical cord (hUC) is the lifeline that connects the fetus to the mother. Hypercoiling of the hUC is associated with pre- and perinatal morbidity and mortality. We investigated the origin of hUC hypercoiling using state-of-the-art imaging and omics approaches. Macroscopic inspection of the hUC revealed the helices to originate from the arteries rather than other components of the hUC. Digital reconstruction of the hUC arteries showed the dynamic alignment of two layers of muscle fibers in the tunica media aligning in opposing directions. We observed that genetically identical twins can be discordant for hUC coiling, excluding genetic, many environmental, and parental origins of hUC coiling. Comparing the transcriptomic and DNA methylation profile of the hUC arteries of four twin pairs with discordant cord coiling, we detected 28 differentially expressed genes, but no differentially methylated CpGs. These genes play a role in vascular development, cell-cell interaction, and axis formation and may account for the increased number of hUC helices. When combined, our results provide a novel framework to understand the origin of hUC helices in fetal development.

Local administration of mesenchymal stromal cells is safe and modulates the immune compartment in ulcerative proctitis.

BACKGROUNDDue to their immunoregulatory and tissue regenerative features, mesenchymal stromal cells (MSCs) are a promising novel tool for the management of ulcerative proctitis (UP). Here we report on a phase IIa clinical study that evaluated the impact of local MSC therapy on UP.METHODSThirteen refractory UP patients, with an endoscopic Mayo score (EMS) of 2 or 3, were included. Seven patients received 20-40 million allogeneic MSCs (cohort 1), while 6 patients received 40-80 million MSCs (cohort 2). Adverse events (AEs) were assessed at baseline and on weeks 2, 6, 12, and 24. Clinical, endoscopic, and biochemical parameters were assessed at baseline and on weeks 2 and 6. Furthermore, we evaluated the engraftment of MSCs, the presence of donor-specific human leukocyte antigen (HLA) antibodies (DSAs), and we determined the impact of MSC therapy on the local immune compartment.RESULTSNo serious AEs were observed. The clinical Mayo score was significantly improved on weeks 2 and 6, and the EMS was significantly improved on week 6, compared with baseline. On week 6, donor MSCs were still detectable in rectal biopsies from 4 of 9 patients and DSAs against both HLA class I and class II were found. Mass cytometry showed a reduction in activated CD8+ T cells and CD16+ monocytes and an enrichment in mononuclear phagocytes and natural killer cells in biopsies after local MSC therapy.CONCLUSIONLocal administration of allogeneic MSCs is safe, tolerable, and feasible for treatment of refractory UP and shows encouraging signs of clinical efficacy and modulation of local immune responses. This sets the stage for larger clinical trials.TRIAL REGISTRATIONEU Clinical Trials Register (EudraCT, 2017-003524-75) and the Dutch Trial Register (NTR7205).FUNDINGECCO grant 2020.

Mesenchymal stromal cells as a tool to unravel the developmental origins of disease.

The intrauterine environment can induce alterations of the epigenome that have a lasting impact on disease risk. Current human studies in the field focus on a single epigenetic mark, DNA methylation, measured in blood. For in-depth mechanistic insight into the developmental origins of disease, it will be crucial to consider innovative tissue types. Mesenchymal stromal cells (MSCs) may serve as a novel tool to investigate the full epigenome beyond DNA methylation, to explore other omics levels, and to perform functional assays. Moreover, MSCs can be differentiated into multiple cell types and thereby mimic otherwise inaccessible cell types. A first wave of studies supports the potential of MSCs and illustrates how the innovative use of this cell type may be incorporated in birth cohorts.

Cytokine Mixtures Mimicking the Local Milieu in Patients with Inflammatory Bowel Disease Impact Phenotype and Function of Mesenchymal Stromal Cells.

Locally applied mesenchymal stromal cells (MSCs) have the capacity to promote the healing of perianal fistulas in Crohn's disease (CD) and are under clinical development for the treatment of proctitis in ulcerative colitis (UC). Despite these clinical advances, the mechanism of action of local MSC therapy in inflammatory bowel disease (IBD) is largely unknown. We hypothesized that the local cytokine environment in IBD patients affects the immunomodulatory properties of MSCs. To evaluate this, 11 cytokines were analyzed in inflamed tissues obtained from CD and UC patients. Based on the identified cytokine profiles 4 distinct cytokine mixtures that mimic various inflammatory IBD environments were established. Next, MSCs were cultured in the presence of either of these 4 cytokine mixtures after which the expression of immunomodulatory and tissue regenerative molecules and the capacity of MSCs to modulate T-cell proliferation and dendritic cell (DC) differentiation were assessed. Our data show that MSCs respond, in a cytokine-specific manner, by upregulation of immunomodulatory and tissue regenerative molecules, including cyclooxygenase-2, indoleamine 2,3-dioxygenase, and transforming growth factor-ß1. Functional studies indicate that MSCs exposed to a cytokine profile mimicking one of the 2 UC cytokine milieus were less effective in inhibition of DC differentiation. In conclusion, our data indicate that cytokine mixes mimicking the local cytokine milieus of inflamed UC colonic or CD fistulas tissues can differentially affect the immunomodulatory and tissue regenerative characteristics of MSCs. These data support the hypothesis that the local intestinal cytokine milieu serves as a critical factor in the efficacy of local MSC treatment.

Hematopoietic stem and progenitor cells are differentially mobilized depending on the duration of Flt3-ligand administration.

BACKGROUND: Flt3-ligand is a cytokine that induces relatively slow mobilization of hematopoietic cells in animals and humans in vivo. This provides a time-frame to study hematopoietic stem and progenitor cell migration kinetics in detail. DESIGN AND METHODS: Mice were injected with Flt3-ligand (10 microg/day, intraperitoneally) for 3, 5, 7 and 10 days. Mobilization of hematopoietic stem and progenitor cells was studied using colony-forming-unit granulocyte/monocyte and cobblestone-area-forming-cell assays. The radioprotective capacity of mobilized peripheral blood mononuclear cells was studied by transplantation of 1.5 x 10(6) Flt3-ligand-mobilized peripheral blood mononuclear cells into lethally irradiated (9.5 Gy) recipients. RESULTS: Hematopoietic progenitor cell mobilization was detected from day 3 onwards and prolonged administration of Flt3-ligand produced a steady increase in mobilized progenitor cells. Compared to Flt3-ligand administration for 5 days, the administration of Flt3-ligand for 10 days led to a 5.5-fold increase in cobblestone-area-forming cells at week 4 and a 5.0-fold increase at week 5. Furthermore, transplantation of peripheral blood mononuclear cells mobilized by 5 days of Flt3-ligand administration did not radioprotect lethally irradiated recipients, whereas peripheral blood mononuclear cells mobilized by 10 days of Flt3-Ligand administration did provide 100% radioprotection of the recipients with significant multilineage donor chimerism. Compared to the administration of Flt3-ligand or interleukin-8 alone, co-administration of interleukin-8 and Flt3-ligand led to synergistic enhancement of hematopoietic stem and progenitor cell mobilization on days 3 and 5. CONCLUSIONS: These results indicate that hematopoietic stem and progenitor cells show different mobilization kinetics in response to Flt3-ligand, resulting in preferential mobilization of hematopoietic progenitor cells at day 5, followed by hematopoietic stem cell mobilization at day 10.

Osteosarcoma originates from mesenchymal stem cells in consequence of aneuploidization and genomic loss of Cdkn2.

High-grade osteosarcoma is characterized by extensive genetic instability, thereby hampering the identification of causative gene mutations and understanding of the underlying pathological processes. It lacks a benign precursor lesion and reports on associations with hereditary predisposition or germline mutations are uncommon, despite the early age of onset. Here we demonstrate a novel comprehensive approach for the study of premalignant stages of osteosarcoma development in a murine mesenchymal stem cell (MSC) system that formed osteosarcomas upon grafting. By parallel functional and phenotypic analysis of normal MSCs, transformed MSCs and derived osteosarcoma cells, we provide substantial evidence for a MSC origin of osteosarcoma. In a stepwise approach, using COBRA-FISH karyotyping and array CGH in different passages of MSCs, we identified aneuploidization, translocations and homozygous loss of the cdkn2 region as the key mediators of MSC malignant transformation. We then identified CDKN2A/p16 protein expression in 88 osteosarcoma patients as a sensitive prognostic marker, thereby bridging the murine MSCs model to human osteosarcoma. Moreover, occasional reports in patients mention osteosarcoma formation following bone marrow transplantation for an unrelated malignancy. Our findings suggest a possible hazard for the clinical use of MSCs; however, they also offer new opportunities to study early genetic events in osteosarcoma genesis and, more importantly, to modulate these events and record the effect on tumour progression. This could be instrumental for the identification of novel therapeutic strategies, since the success of the current therapies has reached a plateau phase.

Cytokine-induced hematopoietic stem and progenitor cell mobilization: unraveling interactions between stem cells and their niche.

Peripheral blood hematopoietic stem and progenitor cells (HSPCs), mobilized by granulocyte colony-stimulating factor, are widely used as a source for both autologous and allogeneic stem cell transplantation. The use of mobilized HSPCs has several advantages over traditional bone marrow-derived HSPCs, including a less invasive harvesting process for the donor, higher HSPC yields, and faster hematopoietic reconstitution in the recipient. For years, the mechanisms by which cytokines and other agents mobilize HSPCs from the bone marrow were not fully understood. The field of stem cell mobilization research has advanced significantly over the past decade, with major breakthroughs in the elucidation of the complex mechanisms that underlie stem cell mobilization. In this review, we provide an overview of the events that underlie HSPC mobilization and address the relevant cellular and molecular components of the bone marrow niche. Furthermore, current and future mobilizing agents will be discussed.

Treating Ischemically Damaged Porcine Kidneys with Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stromal Cells During Ex Vivo Normothermic Machine Perfusion.

Pretransplant normothermic machine perfusion (NMP) of donor kidneys offers the unique opportunity to perform active interventions to an isolated renal graft before transplantation. There is increasing evidence that mesenchymal stromal cells (MSCs) could have a paracrine/endocrine regenerative effect on ischemia-reperfusion injury. The purpose of this study was to determine which cytokines are secreted by MSCs during NMP of a porcine kidney. Viable porcine kidneys and autologous whole blood were obtained from a slaughterhouse. Warm ischemia time was standardized at 20 min and subsequent hypothermic machine perfusion was performed during 2-3 h. Thereafter, kidneys were machine perfused at 37°C during 7 h. After 1 h of NMP, 0, 107 cultured human adipose tissue-derived MSCs, or 107 cultured bone marrow-derived MSCs were added (n = 5 per group). In a fourth experimental group, 7-h NMP was performed with 107 adipose tissue-derived MSCs, without a kidney in the circuit. Kidneys perfused with MSCs showed lower lactate dehydrogenase and neutrophil gelatinase-associated lipocalin levels in comparison with the control group. Also, elevated levels of human hepatocyte growth factor, interleukin (IL)-6, and IL-8 were found in the perfusate of the groups perfused with MSCs compared to the control groups. This study suggests that MSCs, in contact with an injured kidney during NMP, could lead to lower levels of injury markers and induce the release of immunomodulatory cytokines.

Lymphoproliferative Disease in the Rectum 4 Years After Local Mesenchymal Stromal Cell Therapy for Refractory Perianal Crohn's Fistulas: A Case Report.

Mesenchymal stromal cell [MSC] therapy is a new treatment for perianal fistulas in Crohn's disease. Although MSC therapy shows a favourable safety profile, long-term safety data are limited. We detected an Epstein Barr virus [EBV]-associated B cell lymphoproliferative lesion in the rectum of a patient 4 years after local administration of MSCs for his perianal fistulas. To investigate whether MSC therapy contributed to the development of this lymphoproliferative disease, we analyzed the possibility of EBV transfer via the MSC product and the persistence of MSCs in the lymphoproliferative lesion using short tandem repeat analysis.

CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells.

BACKGROUND: CD97 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family of adhesion receptors and is broadly expressed on hematopoietic cells. The aim of this study was to investigate the expression of CD97 on hematopoietic stem- and progenitor cells (HSC/HPC). DESIGN AND METHODS: CD97 expression on hematopoietic stem- and progenitor cells was studied in BALB/c, C57BL/6 and DBA/1 mice using flow cytometry. Functional hematopoietic stem- and progenitor cell characteristics were investigated in vitro and in vivo by progenitor cell assays, cobblestone area forming cell assays and bone marrow cell transplantation. RESULTS: Analysis of CD97 expression on murine bone marrow cells showed three major populations i.e. CD97(HI), CD97(INT) and CD97(NEG) cells. Functional studies revealed that radioprotective capacity and cobblestone area forming cell day 28-35 activity resides in the CD97(INT) bone marrow cell fraction while CFU-GM colony-forming capacity mainly resides in the CD97(NEG) population in all strains. In C57BL/6 and DBA/1 mice CD97(NEG) and CD97(HI) bone marrow cells show hematopoietic stem cell characteristics as well. Further functional analysis of BALB/c CD97(INT) bone marrow cells revealed that c-Kit(HI)CD97(INT) bone marrow cells exhibit HSC activity and are 1.5-fold enriched for cobblestone area forming cell-day 35 activity compared to c-Kit(HI) bone marrow cells. Moreover, phenotypical analysis showed that BALB/c and C57BL/6 HSC are CD97(INT), while DBA/1 HSC are CD97(HI). CONCLUSIONS: CD97 is differentially expressed on hematopoietic stem cells and hematopoietic progenitor cells. Committed progenitor cell activity is largely comprised in the CD97(NEG) fraction, while the CD97(INT) population contains hematopoietic stem cell activity. In BALB/c mice, CD97 expression can be applied to almost completely separate colony-forming cells and cells exhibiting radioprotective capacity. In addition we propose that the CD97(INT)c-Kit(HI) phenotype allows simple and rapid purification of murine hematopoietic stem cells.