Bärbel Brunswig-Spickenheier

Radiation Rescue: Mesenchymal Stromal Cells Protect from Lethal Irradiation

Background Successful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals. Methods and Findings Mouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals. Conclusions Our data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure.

Limited Immune-Modulating Activity of Porcine Mesenchymal Stromal Cells Abolishes Their Protective Efficacy in Acute Kidney Injury

We demonstrated previously that administration of mesenchymal stromal cells (MSCs) after renal ischemia/reperfusion injury (IRI) in rats protected renal function and hastened repair through complex paracrine mechanisms. Here we investigated kidney-protective actions of MSCs in a porcine IRI model that may have relevance to human acute kidney injury (AKI). Groups of female pigs with bilateral IRI were infused with autologous or male allogeneic MSCs. No acute or late complications were observed, but unexpectedly, MSC therapy also had no beneficial effects on kidney function and histology. In vitro, we demonstrated substantial functional and phenotypic overlaps between rodent, human, and porcine MSCs, all of which exhibited trilineage differentiation, characteristic antigen profiles, and secretion of renoprotective vascular endothelial growth factor (VEGF)-A and insulin-like growth factor-1 (IGF-1). However, in striking contrast to human MSCs, porcine MSCs failed to inhibit the mixed lymphocyte reaction (MLR) and induced robust production of proinflammatory interleukin-6 (IL-6). In summary, in contrast to rodent models, treatment of porcine IRI with MSCs was not kidney-protective. This, we conclude, is due to the fact that porcine MSCs exert inadequate immune-modulating effects, further demonstrating that successful therapy of IRI with MSCs critically depends on their anti-inflammatory actions. As a consequence, treatment of AKI with MSCs is not informative regarding the investigation of the underlying mechanisms in this large animal model. We expect, however, that the treatment of human IRI of the kidney with immune-modulating MSCs will be as effective as in rodent models.

HUMAN SEMINAL FLUID CONTAINS SIGNIFICANT QUANTITIES OF PRORENIN : ITS CORRELATION WITH THE SPERM DENSITY

The relevance of the tissue prorenin-renin-angiotensin system (PRAS) to male reproduction has been suggested by several investigators in the past. Although the presence of angiotensin converting enzyme in semen has been demonstrated, unequivocal evidence for the presence of prorenin and renin in the semen is not yet available. We have used a specific immunoradiometric assay based on an antibody directed against the pro-segment of the prorenin molecule to demonstrate that significant quantities of prorenin are present in human semen samples. Although semen is a rich source of proteases and protease inhibitors, the assay used by us, unlike the usual enzymatic renin assay, is not affected by such proteases, and their inhibitors. Furthermore, Western blotting data clearly demonstrated that prorenin is present in semen as a 48 kDa protein. In a majority of semen samples, the prorenin content was found to be several fold greater than that measured in EDTA-plasma samples. Interestingly, the level of prorenin was found to be directly proportional to the sperm density in semen samples. Our results suggest that seminal prorenin is produced locally within the male reproductive system, although its exact origin is yet to be defined, that a complete prorenin-renin-angiotensin system exists in human semen and that this system may be relevant to sperm function.

Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.

The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.

Platelet lysate suppresses the expression of lipocalin-type prostaglandin D2 synthase that positively controls adipogenic differentiation of human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) have been shown to display a considerable therapeutic potential in cellular therapies. However, harmful adipogenic maldifferentiation of transplanted MSCs may seriously threaten the success of this therapeutic approach. We have previously demonstrated that using platelet lysate (PL) instead of widely used fetal calf serum (FCS) diminished lipid accumulation in adipogenically stimulated human MSCs and identified, among others, lipocalin-type prostaglandin D2 synthase (L-PGDS) as a gene suppressed in PL-supplemented MSCs. Here, we investigated the role of PL and putatively pro-adipogenic L-PGDS in human MSC adipogenesis. Next to strongly reduced levels of L-PGDS we show that PL-supplemented MSCs display markedly decreased expression of adipogenic master regulators and differentiation markers, both before and after induction of adipocyte differentiation. The low adipogenic differentiation capability of PL-supplemented MSCs could be partially restored by exogenous addition of L-PGDS protein. Conversely, siRNA-mediated downregulation of L-PGDS in FCS-supplemented MSCs profoundly reduced adipocyte differentiation. In contrast, inhibiting endogenous prostaglandin synthesis by aspirin did not reduce differentiation, suggesting that a mechanism such as lipid shuttling but not the prostaglandin D2 synthase activity of L-PGDS is critical for adipogenesis. Our data demonstrate that L-PGDS is a novel pro-adipogenic factor in human MSCs which might be of relevance in adipocyte metabolism and disease. L-PGDS gene expression is a potential quality marker for human MSCs, as it might predict unwanted adipogenic differentiation after MSC transplantation.

Mesenchymal Stromal Cell‐Derived Extracellular Vesicles Provide Long‐Term Survival After Total Body Irradiation Without Additional Hematopoietic Stem Cell Support

The therapeutic effect of mesenchymal stromal cells (MSC) in tissue regeneration is based mainly on the secretion of bioactive molecules. Here, we report that the radioprotective effect of mouse bone marrow derived mesenchymal stromal cells (mMSC) can be attributed to extracellular vesicles (EV) released from mMSC. The transplantation of mMSC‐derived EV into lethally irradiated mice resulted in long‐term survival but no improvement in short‐term reconstitution of the recipients. Importantly, the radiation rescue was efficient without additional hematopoietic support. In vitro we show a protection by EV of irradiated hematopoietic stem cells but not progenitor cells using stroma‐cell cultures and colony‐forming assays. After systemic infusion into lethally irradiated recipients, labeled EV traveled freely through the body reaching the bone marrow within 2 hours. We further show that long‐term repopulating Sca‐1 positive and c‐kit low‐positive stem cells were directly targeted by EV leading to long‐term survival. Collectively, our data suggest EV as an effective first‐line treatment to combat radiation‐induced hematopoietic failure which might also be helpful in alleviating myelosuppression due to chemotherapy and toxic drug reaction. We suggest the infusion of MSC‐derived EV as efficient and immediate treatment option after irradiation injuries. Stem Cells 2017;35:2379–2389

The effect of microtubule-disrupting drugs on morphology, progesterone and prorenin secretion of bovine cultured ovarian theca cells.

The effect of three microtubule-disrupting drugs (vinblastine, colchicine and nocodazole) on basal and LH-stimulated secretion of progesterone and prorenin by bovine theca cells was studied. Microtubules were visualized immunocytochemically using a monoclonal antibody against the alpha-subunit of tubulin and a secondary antibody conjugated with rhodamine. Progesterone and prorenin secretion were detected by adequate radioimmunoassays. Theca cells treated with LH alone or with a combination of LH, colchicine and vinblastine, showed round shape and disorganization of microtubules which were more obvious than after treatment with the two disrupting drugs alone. Nocodazole or LH treatment alone resulted in the formation of numerous cell processes, conspicuously different in shape from those in the controls. All three drugs increased basal progesterone secretion independently on the dose, but were without effect on basal prorenin secretion. LH-stimulated progesterone secretion was significantly suppressed by each of the three drugs while LH-stimulated prorenin secretion was decreased only by high doses of vinblastine and nocodazole. Colchicine in all three doses used did not exert any significant effect. The results obtained suggest that microtubules are involved in progesterone but not in prorenin secretion.

PARACRINE REGULATION OF THE BOVINE OVARIAN PRORENIN-RENIN-ANGIOTENSIN­ SYSTEM

In the bovine ovary, prorenin production by theca cells is known to be regulated by LH. In the present study the aim was to evaluate whether LH-stimulated prorenin production could be further modulated by intraovarian factors in vitro. Theca cells were isolated from bovine ovaries by enzymatic dispersion, purified over Percoll gradient and cultured under serum-free conditions with LH/8Br-cAMP in the absence or presence of different steroids and growth factors and the amount of prorenin secreted into the medium was measured. None of the steroids used (androstendione, estradiol, progesterone) influenced the basal or LH-stimulated prorenin production. In contrast, cytokines and growth factors, like TNFα, TGFα, TGFβ and bFGF proved to be important regulators of prorenin synthesis. Whereas TNFα, TGFα and bFGF significantly reduced the LH-and 8Br-cAMP-induced prorenin synthesis at a site distal to cAMP formation, addition of TGFβ led to a further increase in the amount of prorenin secreted into the medium. None of the agonists had an influence on prorenin production by itself. The observed effects of cytokines and growth factors seemed to be confined to prorenin production only, since cell number, cell viability and steroidogenic response were not at all influenced by the agonists. We conclude that, although LH appears to be the primary regulator of ovarian prorenin production, several paracrine/autocrine intraovarian factors may be involved in “finely tuning” the secretion of prorenin, which is necessary for maintaining the differentiated state of the follicle.

Mesenchymal stromal cells protect from consequences of HSCT-transplantation preparatory irradiation: insights into possible mechanisms

Ionizing irradiation is widely used as conditioning therapy in bone marrow (BM) transplantation. High-dose radiation treatment induces profound tissue damage, especially, of hematopoietic stem cells and progenitor cells. Efforts to improve clinical outcomes postirradiation are focused on the hematopoietic stem cell niche. Mesenchymal stromal cells (MSCs) represent an integrative part of the BM stromal microenvironment. When co-transplanted with HSC, MSCs augment hematopoietic recovery after chemoor radiotherapy. The aim of our study was to evaluate essential biological parameters of MSCs, with respect to their lineage-specific differentiation capacity, in vivo survival rates, as well as their ability to rescue lethally irradiated hosts. Materials and Methods. In vitro differentiation of human BM-derived MSCs (hMSCs) for hematopoietic (HSC) and endothelial cells (EC) was studied by reverse transcription-quantitative PCR (RT-qPCR) of lineage-specific surface markers and other proteins. To test in vivo ability of murine MSCs to rescue lethally irradiated (9.5 Gy) mice, the animals were transplanted with eGFP-marked murine MSCs (mMSCs). Long-term donor chimerism was assessed in blood, BM and thymus using CD45.2 and Y chromosome markers. A microarray analysis of bone marrow cells from MSC-transplanted animals was also performed, in order to compare their gene expression profiles to appropriate controls.