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.

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.

HIF signaling in osteoblast-lineage cells promotes systemic breast cancer growth and metastasis in mice.

Bone metastasis involves dynamic interplay between tumor cells and the local stromal environment. In bones, local hypoxia and activation of the hypoxia-inducible factor (HIF)-1α in osteoblasts are essential to maintain skeletal homeostasis. However, the role of osteoblast-specific HIF signaling in cancer metastasis is unknown. Here, we show that osteoprogenitor cells (OPCs) are located in hypoxic niches in the bone marrow and that activation of HIF signaling in these cells increases bone mass and favors breast cancer metastasis to bone locally. Remarkably, HIF signaling in osteoblast-lineage cells also promotes breast cancer growth and dissemination remotely, in the lungs and in other tissues distant from bones. Mechanistically, we found that activation of HIF signaling in OPCs increases blood levels of the chemokine C-X-C motif ligand 12 (CXCL12), which leads to a systemic increase of breast cancer cell proliferation and dissemination through direct activation of the CXCR4 receptor. Hence, our data reveal a previously unrecognized role of the hypoxic osteogenic niche in promoting tumorigenesis beyond the local bone microenvironment. They also support the concept that the skeleton is an important regulator of the systemic tumor environment.

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.

Mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease?

COPD is characterised by tissue destruction and inflammation. Given the lack of curative treatments and the progressive nature of the disease, new treatments for COPD are highly relevant. In vitro cell culture and animal studies have demonstrated that mesenchymal stromal cells (MSCs) have the capacity to modify immune responses and to enhance tissue repair. These properties of MSCs provided a rationale to investigate their potential for treatment of a variety of diseases, including COPD. Preclinical models support the hypothesis that MSCs may have clinical efficacy in COPD. However, although clinical trials have demonstrated the safety of MSC treatment, thus far they have not provided evidence for MSC efficacy in the treatment of COPD. In this review, we discuss the rationale for MSC-based cell therapy in COPD, the main findings from in vitro and in vivo preclinical COPD model studies, clinical trials in patients with COPD and directions for further research.

Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils.

Expansion of myeloid cells associated with solid tumor development is a key contributor to neoplastic progression. Despite their clinical relevance, the mechanisms controlling myeloid cell production and activity in cancer remains poorly understood. Using a multistage mouse model of breast cancer, we show that production of atypical T cell-suppressive neutrophils occurs during early tumor progression, at the onset of malignant conversion, and that these cells preferentially accumulate in peripheral tissues but not in the primary tumor. Production of these cells results from activation of a myeloid differentiation program in bone marrow (BM) by a novel mechanism in which tumor-derived granulocyte-colony stimulating factor (G-CSF) directs expansion and differentiation of hematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. Chronic skewing of myeloid production occurred in parallel to a decrease in erythropoiesis in BM in mice with progressive disease. Significantly, we reveal that prolonged G-CSF stimulation is both necessary and sufficient for the distinguishing characteristics of tumor-induced immunosuppressive neutrophils. These results demonstrate that prolonged G-CSF may be responsible for both the development and activity of immunosuppressive neutrophils in cancer.

Autophagy Proteins ATG5 and ATG7 Are Essential for the Maintenance of Human CD34(+) Hematopoietic Stem-Progenitor Cells.

Autophagy is a highly regulated catabolic process that involves sequestration and lysosomal degradation of cytosolic components such as damaged organelles and misfolded proteins. While autophagy can be considered to be a general cellular housekeeping process, it has become clear that it may also play cell type-dependent functional roles. In this study, we analyzed the functional importance of autophagy in human hematopoietic stem/progenitor cells (HSPCs), and how this is regulated during differentiation. Western blot-based analysis of LC3-II and p62 levels, as well as flow cytometry-based autophagic vesicle quantification, demonstrated that umbilical cord blood-derived CD34(+) /CD38(-) immature hematopoietic progenitors show a higher autophagic flux than CD34(+) /CD38(+) progenitors and more differentiated myeloid and erythroid cells. This high autophagic flux was critical for maintaining stem and progenitor function since knockdown of autophagy genes ATG5 or ATG7 resulted in reduced HSPC frequencies in vitro as well as in vivo. The reduction in HSPCs was not due to impaired differentiation, but at least in part due to reduced cell cycle progression and increased apoptosis. This is accompanied by increased expression of p53, proapoptotic genes BAX and PUMA, and the cell cycle inhibitor p21, as well as increased levels of cleaved caspase-3 and reactive oxygen species. Taken together, our data demonstrate that autophagy is an important regulatory mechanism for human HSCs and their progeny, reducing cellular stress and promoting survival. Stem Cells 2016;34:1651-1663.

Activated Gs signaling in osteoblastic cells alters the hematopoietic stem cell niche in mice.

Adult hematopoiesis occurs primarily in the BM space where hematopoietic cells interact with stromal niche cells. Despite this close association, little is known about the specific roles of osteoblastic lineage cells (OBCs) in maintaining hematopoietic stem cells (HSCs), and how conditions affecting bone formation influence HSC function. Here we use a transgenic mouse model with the ColI(2.3) promoter driving a ligand-independent, constitutively active 5HT4 serotonin receptor (Rs1) to address how the massive increase in trabecular bone formation resulting from increased G(s) signaling in OBCs impacts HSC function and blood production. Rs1 mice display fibrous dysplasia, BM aplasia, progressive loss of HSC numbers, and impaired megakaryocyte/erythrocyte development with defective recovery after hematopoietic injury. These hematopoietic defects develop without compensatory extramedullary hematopoiesis, and the loss of HSCs occurs despite a paradoxical expansion of stromal niche cells with putative HSC-supportive activity (ie, endothelial, mesenchymal, and osteoblastic cells). However, Rs1-expressing OBCs show decreased expression of key HSC-supportive factors and impaired ability to maintain HSCs. Our findings indicate that long-term activation of G(s) signaling in OBCs leads to contextual changes in the BM niche that adversely affect HSC maintenance and blood homeostasis.

CD4 memory T cells survive and proliferate but fail to differentiate in the absence of CD40.

Secondary T cell responses are enhanced because of an expansion in numbers of antigen-specific (memory) cells. Using major histocompatibility complex class II tetramers we have tracked peptide-specific endogenous (non-T cell receptor transgenic) CD4 memory T cells in normal and in costimulation-deficient mice. CD4 memory T cells were detectable after immunization for more than 200 days, although decay was apparent. Memory cells generated in CD40 knockout mice by immunization with peptide-pulsed wild-type dendritic cells survived in the absence of CD40 and proliferated when boosted with peptide (plus adjuvant) in a CD40-independent fashion. However, differentiation of the memory cells into cytokine-producing effector cells did not occur in the absence of CD40. The data indicate that memory cells can be generated without passing through the effector cell stage.

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.

Tumor rejection induced by CD70-mediated quantitative and qualitative effects on effector CD8+ T cell formation.

In vivo priming of antigen-specific CD8+ T cells results in their expansion and differentiation into effector T cells followed by contraction into a memory T cell population that can be maintained for life. Recent evidence suggests that after initial antigenic stimulation, the magnitude and kinetics of the CD8+ T cell response are programmed. However, it is unclear to what extent CD8+ T cell instruction in vivo is modulated by costimulatory signals. Here, we demonstrate that constitutive ligation of the tumor necrosis factor receptor family member CD27 by its ligand CD70 quantitatively augments CD8+ T cell responses to influenza virus infection and EL-4 tumor challenge in vivo by incrementing initial expansion and maintaining higher numbers of antigen-specific T cells in the memory phase. Concomitantly, the quality of antigen-specific T cells improved as evidenced by increased interferon (IFN)-gamma production and a greater cytotoxic potential on a per cell basis. As an apparent consequence, the superior effector T cell formation induced by CD70 protected against a lethal dose of poorly immunogenic EL4 tumor cells in a CD8+ T cell- and IFN-gamma-dependent manner. Thus, CD70 costimulation enhances both the expansion and per cell activity of antigen-specific CD8+ T cells.

Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures.

To introduce a functional vascular network into tissue-engineered bone equivalents, human endothelial colony forming cells (ECFCs) and multipotent mesenchymal stromal cells (MSCs) can be cocultured. Here, we studied the impact of donor variation of human bone marrow-derived MSCs and cord blood-derived ECFCs on vasculogenesis and osteogenesis using a 3D in vitro coculture model. Further, to make the step towards cocultures consisting of cells derived from a single donor, we tested how induced pluripotent stem cell (iPSC)-derived human endothelial cells (iECs) performed in coculture models. Cocultures with varying combinations of human donors of MSCs, ECFCs, or iECs were prepared in Matrigel. The constructs were cultured in an osteogenic differentiation medium. Following a 10-day culture period, the length of the prevascular structures and osteogenic differentiation were evaluated for up to 21 days of culture. The particular combination of MSC and ECFC donors influenced the vasculogenic properties significantly and induced variation in osteogenic potential. In addition, the use of iECs in the cocultures resulted in prevascular structure formation in osteogenically differentiated constructs. Together, these results showed that close attention to the source of primary cells, such as ECFCs and MSCs, is critical to address variability in vasculogenic and osteogenic potential. The 3D coculture model appeared to successfully generate prevascularized constructs and were sufficient in exceeding the ~200 µm diffusion limit. In addition, iPSC-derived cell lineages may decrease variability by providing a larger and potentially more uniform source of cells for future preclinical and clinical applications.

Unraveling mechanisms of mesenchymal stromal cell-mediated immunomodulation through patient monitoring and product characterization.

Mesenchymal stromal cells (MSCs) are increasingly used in the treatment of a variety of clinical conditions and to modulate immune responses in conditions related to auto-/alloimmunity, including graft-versus-host disease (GvHD). Although pilot data are promising, treatment responses have been highly variable, and further development of this as a therapeutic modality depends on increased insight into the properties of clinical MSC products and on understanding the mechanisms underlying responses in patients. Here we review the mechanisms that possibly underlie the capacity of MSCs to treat auto-/alloimmunity, and describe how patient monitoring can help to identify the in vivo mechanisms of action in the treatment of GvHD. Since MSCs used in the clinic originate from various donors and from a heterogeneous population of cells, we will also discuss recent insights into MSC heterogeneity and their implications for clinical MSC products. Finally, we describe a framework to improve our understanding of the efficacy and working mechanism of MSCs, which involves patient monitoring and more extensive characterization of the heterogeneity within and between different MSC preparations.

The human and murine hematopoietic stem cell niches: are they comparable?

Hematopoietic stem cells (HSCs) reside in specific niches that provide various instructive cues that regulate HSC self-renewal and their development into all mature cells of the peripheral blood. Progress in this research field has largely been guided by mouse studies. However, parallel studies with human subjects, tissues, and cells, in combination with xenotransplantation experiments in immunodeficient mice, have contributed to our increased understanding of the human HSC niche. Here, we summarize our current knowledge of the various specialized subsets of both stromal and hematopoietic cells that support HSCs through cell-cell interactions and secreted factors, and the many parallels between the murine and human HSC niches. Furthermore, we discuss recent technological advances that are likely to improve our understanding of the human HSC niche, a better understanding of which may allow further identification of unique molecular and cellular pathways in the HSC niche. This information may help to further improve the outcome of HSC transplantation and refine the treatment of hematopoietic diseases.

Normal and leukemic stem cell niches: insights and therapeutic opportunities.

Hematopoietic stem cells (HSCs) rely on instructive cues from the bone marrow (BM) niche to maintain their quiescence and adapt blood production to the organism's needs. Alterations in the BM niche are commonly observed in blood malignancies and directly contribute to the aberrant function of disease-initiating leukemic stem cells (LSCs). Here, we review recent insights into the cellular and molecular determinants of the normal HSC niche and describe how genetic changes in stromal cells and leukemia-induced BM niche remodeling contribute to blood malignancies. Moreover, we discuss how these findings can be applied to non-cell-autonomous therapies targeting the LSC niche.

In Vitro Evaluation of Spider Silk Meshes as a Potential Biomaterial for Bladder Reconstruction.

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we investigate the application of spider major ampullate gland-derived dragline silk from the Nephila edulis spider, a natural biomaterial with outstanding mechanical properties and a slow degradation rate, as a potential scaffold for bladder reconstruction by studying the cellular response of primary bladder cells to this biomaterial. We demonstrate that spider silk without any additional biological coating supports adhesion and growth of primary human urothelial cells (HUCs), which are multipotent bladder cells able to differentiate into the various epithelial layers of the bladder. HUCs cultured on spider silk did not show significant changes in the expression of various epithelial-to-mesenchymal transition and fibrosis associated genes, and demonstrated only slight reduction in the expression of adhesion and cellular differentiation genes. Furthermore, flow cytometric analysis showed that most of the silk-exposed HUCs maintain an undifferentiated immunophenotype. These results demonstrate that spider silk from the Nephila edulis spider supports adhesion, survival and growth of HUCs without significantly altering their cellular properties making this type of material a suitable candidate for being tested in pre-clinical models for bladder reconstruction.

Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche.

Multipotent stromal cells (MSCs) and their osteoblastic lineage cell (OBC) derivatives are part of the bone marrow (BM) niche and contribute to hematopoietic stem cell (HSC) maintenance. Here, we show that myeloproliferative neoplasia (MPN) progressively remodels the endosteal BM niche into a self-reinforcing leukemic niche that impairs normal hematopoiesis, favors leukemic stem cell (LSC) function, and contributes to BM fibrosis. We show that leukemic myeloid cells stimulate MSCs to overproduce functionally altered OBCs, which accumulate in the BM cavity as inflammatory myelofibrotic cells. We identify roles for thrombopoietin, CCL3, and direct cell-cell interactions in driving OBC expansion, and for changes in TGF-ß, Notch, and inflammatory signaling in OBC remodeling. MPN-expanded OBCs, in turn, exhibit decreased expression of many HSC retention factors and severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of MPN-affected patients and prevention of myelofibrosis.

Dynamic expression of the Robo ligand Slit2 in bone marrow cell populations.

The bone marrow (BM) niche is essential for lifelong hematopoietic stem cell (HSC) maintenance, proliferation and differentiation. Several BM cell types, including osteoblast lineage cells (OBC), mesenchymal stem cells (MSC) and endothelial cells (EC) have been implicated in supporting HSC location and function, but the relative importance of these cell types and their secreted ligands remain controversial. We recently found that the cell surface receptors Robo4 and CXCR4 cooperate to localize HSC to BM niches. We hypothesized that Slit2, a putative ligand for Robo4, cooperates with the CXCR4 ligand SDF1 to direct HSC to specific BM niche sites. Here, we have isolated OBC, MSC and EC by flow cytometry and determined their frequency within the bone marrow and the relative mRNA levels of Slit2, SDF1 and Robo4. We found that expression of Slit2 and SDF1 were dynamically regulated in MSC and OBC-like populations following radiation, while Robo4 expression was restricted to EC. Radiation also significantly affected the cellularity and frequency of both the non-adherent and adherent cells within the BM stroma. These data support a physiological role for Slit2 in regulating the dynamic function of Robo-expressing cells within BM niches at steady state and following radiation.

One naive T cell, multiple fates in CD8+ T cell differentiation.

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR-pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8+ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.