Kalvolipidien ja solunulkoisten vesikkelien vaikutus mesenkymaalisten stroomasolujen kykyyn hillitä immunologisia reaktioita

Human mesenchymal stromal cells (hMSCs) are currently used in many advanced cellular therapies. The clinical use of hMSCs requires extensive cell expansion, but the consequences of expansion, especially at the molecular level, are not fully understood. The therapeutic effect of hMSCs is mediated paracrine interactions with immune cells modulating both innate and adaptive immune response. Membrane glycerophospholipids (GPLs) provide precursors for signaling lipids, which modulate cellular functions, including immunological effects. In this thesis, I investigated the effect of the donor’s age and cell doublings on the GPLs, gene expression and microRNA (miRNA) profiles of human bone marrow MSCs (hBM-MSCs). In order to gain more insight into the functional mechanisms of hMSCs, I investigated the extracellular vesicle secretion from human umbilical cord blood derived MSCs, and evaluated their immunosuppressive capacity in vitro as well as their possible immunomodulative and protective effect in kidney ischemia-reperfusion injury in vivo. In this study, I was able to demonstrate that the hBM-MSCs, harvested from 5 young adults and 5 old donors, showed clear compositional changes in their GPL profiles during expansion. Most strikingly, the molar ratio of arachidonic acid (20:4n-6) containing species of phosphatidylcholine and phosphatidylethanolamine accumulated, while the species containing monounsaturated fatty acids decreased during passaging. The lipid changes correlated with the decreased immunosuppressive capacity of hBM-MSCs suggesting a connection between lipid signaling and immunomodulatory functions. Although there were clear alterations in gene expression levels and lipid profiles, the miRNA expression levels were more stable. The expression levels of 37 miRNAs were changed in the old donors group and 36 miRNAs were changed in the young donors group. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs and their predicted target mRNAs, the expression of which was changed, were mainly linked to cell proliferation and senescence. This thesis provides a detailed analysis of molecular changes during MSC expansion. The combination of in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics is essential to understand the complexity of the MSC paracrine mechanisms and functionality.Mesenkymaaliset stroomasolut (MSC) ovat aikuisten kantasoluja, joilla on osoitettu olevan kyky vaimentaa elimistössä käynnistynyttä puolustusreaktiota. Näitä soluja käytetään tällä hetkellä kokeellisena hoitoa esimerkiksi kantasolusiirtoihin liittyvässä käänteishyljintäsairaudessa, missä siirteen valkosolut ovat hyökänneet potilaan omia kudoksia vastaan aiheuttaen monimuotoisia usein vakaviakin iho-, maksa- ja suolisto-oireita. Solujen rasva-aineiden tehtävä ei ole vain toimia solujen kalvorakenteiden rakennuspalikoina tai energiavarastoina. Monet solujen sisäistä ja ulkoisista viesteistä kuljetetaan erilaisten aktiivisten vesiliukoisten rasva-ainemolekyylien välityksellä. Useimmat näistä niin kutsutuista bioaktiivisista lipideistä valmistetaan solukalvojen rasva-aineista. Soluhoitoja varten mesenkymaalisia kantasoluja tulee viljellä keinotekoisissa olosuhteissa elimistön ulkopuolella. Tässä väitöskirjassa tutkittiin solukalvojen rasva-aineiden määräsuhtiessa, geenien ilmentymisessä ja geenien ilmentymistä säätelevien pienten RNA molekyylien (mikroRNA) ilmentymisessä tapahtuvia muutoksia mesenkymaalisten stroomasolujen kasvatuksen ja vanhenemisen aikana. Osoitin tiettyjen monityydyttymättömien rasvahappojen määräsuhteiden muutosten olevan yhteydessä solujen kykyyn hillitä T-solujen jakautumista, merkkinä solujen puolustusreaktiota hillitsevästä ominaisuudesta. Solujen välisessä viestinnässä keskeisessä roolissa ovat pienet kalvorakkulat, vesikkelit. Mesenkymaalisten stroomasolujen on osoitettu vaikuttavan immuunipuolustuksen soluihin vesikkeleitä vapauttamalla. Tässä tutkimuksessa osoitin, että mesenkymaalisten stroomasolujen tuottamilla vesikkeleillä on kyky hillitä tulehdusreaktiota. Mesenkymaalisten stroomasolujen ominaisuksien ja toimintamekanismien tuntemus on välttämätöntä turvallisten ja tehokkaiden soluterapiatuotteiden kehittämiseksi

Metabolism and phospholipid assembly of polyunsaturated fatty acids in human bone marrow mesenchymal stromal cells

High arachidonic acid (20:4n-6) and low n-3 PUFA levels impair the capacity of cultured human bone marrow mesenchymal stromal cells (hBMSCs) to modulate immune functions. The capacity of the hBMSCs to modify PUFA structures was found to be limited. Therefore, different PUFA supplements given to the cells resulted in very different glycerophospholipid (GPL) species profiles and substrate availability for phospholipases, which have preferences for polar head group and acyl chains when liberating PUFA precursors for production of lipid mediators. When supplemented with 20:4n-6, the cells increased prostaglandin E2 secretion. However, they elongated 20:4n-6 to the less active precursor, 22:4n-6, and also incorporated it into triacylglycerols, which may have limited the proinflammatory signaling. The n-3 PUFA precursor, 18:3n-3, had little potency to reduce the GPL 20:4n-6 content, while the eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acid supplements efficiently displaced the 20:4n-6 acyls, and created diverse GPL species substrate pools allowing attenuation of inflammatory signaling.(Jlr) The results emphasize the importance of choosing appropriate PUFA supplements for in vitro hBMSC expansion and suggests that for optimal function they require an exogenous fatty acid source providing 20:5n-3 and 22:6n-3 sufficiently, but 20:4n-6 moderately, which calls for specifically designed optimal PUFA supplements for the cultures.Peer reviewe

The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells from human cord blood

Background aims. Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. Methods. CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. Results. We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. Conclusions. Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.Peer reviewe

Expansion induced microRNA changes in bone marrow mesenchymal stromal cells reveals interplay between immune regulation and cell cycle

Mesenchymal stromal cells (MSC) are currently used in many cell based therapies. Prior to use in therapy, extensive expansion is required. We used microarray profiling to investigate expansion induced miRNA and mRNA expression changes of bone marrow MSCs (BM-MSCs) derived from old and young donors. The expression levels of 36 miRNAs were altered in cells derived from the old and respectively 39 miRNAs were altered in cells derived from young donors. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs, and their predicted target mRNAs, were mainly linked to cell proliferation and senescence. Further qPCR verification showed that the expression of miR-1915-3p, miR-1207, miR-3665, and miR-762 correlated with the expansion time at passage 8. Previously described BM-MSC-specific miRNA fingerprints were also detected but these remained unchanged during expansion. Interestingly, members of well-studied miR-17/92 cluster, involved in cell cycle regulation, aging and also development of immune system, were down regulated specifically in cells from old donors. The role of this cluster in MSC functionality is worth future studies since it links expansion, aging and immune system together.Peer reviewe

Methylome Analysis of Human Bone Marrow MSCs Reveals Extensive Age- and Culture-Induced Changes at Distal Regulatory Elements

Human bone marrow stromal cells, or mesenchymal stem cells (BM-MSCs), need expansion prior to use as cell-based therapies in immunological and tissue repair applications. Aging and expansion of BM-MSCs induce epigenetic changes that can impact therapeutic outcomes. By applying sequencing-based methods, we reveal that the breadth of DNA methylation dynamics associated with aging and expansion is greater than previously reported. Methylation changes are enriched at known distal transcription factor binding sites such as enhancer elements, instead of CpG-rich regions, and are associated with changes in gene expression. From this, we constructed hypo- and hypermethylation-specific regulatory networks, including a sub-network of BM-MSC master regulators and their predicted target genes, and identified putatively disrupted signaling pathways. Our genome-wide analyses provide a broader overview of age- and expansion-induced DNA methylation changes and a better understanding of the extent to which these changes alter gene expression and functionality of human BM-MSCs.</p

Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality

Peer reviewe

Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning

Background: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus. Methods: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. Results: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. Conclusions: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation

Methylome analysis of human bone marrow MSCs reveals extensive age- and culture-induced changes at distal regulatory elements

Abstract Human bone marrow stromal cells, or mesenchymal stem cells (BM-MSCs), need expansion prior to use as cell-based therapies in immunological and tissue repair applications. Aging and expansion of BM-MSCs induce epigenetic changes that can impact therapeutic outcomes. By applying sequencing-based methods, we reveal that the breadth of DNA methylation dynamics associated with aging and expansion is greater than previously reported. Methylation changes are enriched at known distal transcription factor binding sites such as enhancer elements, instead of CpG-rich regions, and are associated with changes in gene expression. From this, we constructed hypo- and hypermethylation-specific regulatory networks, including a sub-network of BM-MSC master regulators and their predicted target genes, and identified putatively disrupted signaling pathways. Our genome-wide analyses provide a broader overview of age- and expansion-induced DNA methylation changes and a better understanding of the extent to which these changes alter gene expression and functionality of human BM-MSCs