Mesenchymal stromal cells and circulating angiogenic cells in vascularization

Vascularization is essential for tissue healing and reduced ability to regrow blood vessels can contribute, for example, to impaired wound healing and bone formation after injury. Generation of functional blood vessels is the key challenge for regenerative medicine and further development of vascular tissue engineering methods are still needed. Mesenchymal stromal/stem cells (MSCs) contribute to tissue healing through their differentiation capacity and by promoting new blood vessel formation. Endothelial progenitor cells (EPCs), which can be isolated from peripheral blood (PB) and cultured in vitro are consistently reported to produce two different types of EPCs. Endothelial colony forming cells (ECFCs) can differentiate into mature endothelial cells and themselves form capillary-like structures, while myeloid angiogenic cells (MACs) increase vascularization in a paracrine manner. Thus, co-cultures of MSCs and PB derived cells may present a potential cell-based tool to enhance angiogenesis in tissue transplants and to promote tissue healing. In this thesis, increased expression of proangiogenic factors was demonstrated in co-cultures of bone marrow (BM) derived MSCs and PB derived mononuclear cells (MNCs) in both basal and osteoblastic culture conditions, while the expression profiles differed between the two conditions. Furthermore, differentiation of functional MACs was observed in the MSC-MNC co-cultures and pericyte-like cells with different marker profiles were discovered within both MSCs and MNCs. In addition, we showed, for the first time, that circulating MACs and hematopoietic pericyte-like cells are found in both patients with traumatic skin defect and in healthy controls. These results suggest that MACs are not only generated in vitro but they do exist in circulation in vivo. To improve the therapeutic competency of MSCs and MNCs, it is important to understand their contribution to neovascularization. Results presented here provide important knowledge on the functions and interactions between MSCs and MNCs and thus improve their potential for future clinical use in regenerative medicine.Mesenkymaaliset kantasolut ja verenkierron angiogeeniset solut verisuonten uudismuodostuksessa Verisuonten uudismuodostus on merkittävässä roolissa kudosvaurioiden korjaamisessa ja heikentyneen verisuonituksen seurauksena voikin olla muun muassa haavan tai luunmurtuman paranemisen hidastuminen. Regeneratiivisen lääketieteen yksi suurimmista haasteista on saada aikaan tehokas ja toimiva verisuonitus, minkä vuoksi tarvitaan menetelmiä, joilla esimerkiksi kudossiirteiden verisuonten uudismuodostusta voitaisiin tehostaa. Mesenkymaaliset kantasolut (eng. MSCs) edistävät kudosten paranemista niin erilaistumalla mesenkymaalisten kudosten eri soluiksi kuin erittämällä esimerkiksi tulehdusreaktiota estäviä sekä verisuonten muodostukseen tarvittavia tekijöitä. Verenkierrosta eristettävä mononukleaarisolupopulaatio (eng. MNCs) puolestaan sisältää kahdenlaisia verisuonten uudismuodostukseen osallistuvia endoteelisolujen esiasteita. Todelliset endoteelisolujen esiastesolut voivat itse muodostaa verisuonimaisia rakenteita, kun taas myeloidisen linjan angiogeeniset esiastesolut (eng. MACs) lisäävät verisuonten muodostusta parakriinisesti. Mesenkymaalisten kantasolujen ja verenkierron solujen yhteisviljelmiä voidaankin mahdollisesti hyödyntää kudosteknologian alalla uudisverisuonituksen tehostamiseksi kudossiirteissä. Tässä väitöskirjassa osoitettiin, että kun MSC-soluja ja verenkierron MNC-soluja viljeltiin yhdessä sekä perusolosuhteissa, että luun muodostusta lisäävissä olosuhteissa, verisuonten muodostukseen tarvittavien tekijöiden ilmentyminen lisääntyi. Lisäksi osoitettiin, että yhteisviljelmässä erilaistui sekä verisuonten muodostukseen tarvittavia perisyyttejä että toiminnallisia MAC-soluja. Näiden tulosten lisäksi MAC-soluja ja perisyyttimäisiä soluja löydettiin traumaattisista ihovaurioista kärsivien potilaiden sekä terveiden luovuttajien verenkierrosta. Nämä tulokset viittaavat siihen, että MAC-soluja ei muodostu vain viljelyolosuhteissa, vaan niitä esiintyy myös verenkierrossa. MSC:jen ja MNC:jen välisten vuorovaikutusten ja niiden merkityksen ymmärtäminen kudosten korjaamisessa ja uusiutumisessa onkin keskeistä, jotta niitä voitaisiin tulevaisuudessa paremmin hyödyntää kliinisessä käytössä esimerkiksi tehostamassa uudisverisuonitusta kudossiirteessä

Angiogenic potential of human mesenchymal stromal cell and circulating mononuclear cell cocultures is reflected in the expression profiles of proangiogenic factors leading to endothelial cell and pericyte differentiation.

Endothelial progenitors found among the peripheral blood (PB) mononuclear cells (MNCs) are interesting cells for their angiogenic properties. Mesenchymal stromal cells (MSCs) in turn can produce proangiogenic factors as well as differentiate into mural pericytes, making MSCs and MNCs an attractive coculture setup for regenerative medicine. In this study, human bone marrow-derived MSCs and PB-derived MNCs were cocultured in basal or osteoblastic medium without exogenously supplied growth factors to demonstrate endothelial cell, pericyte and osteoblastic differentiation. The expression levels of various proangiogenic factors, as well as endothelial cell, pericyte and osteoblast markers in cocultures were determined by quantitative polymerase chain reaction. Immunocytochemistry for vascular endothelial growth factor receptor-1 and α-smooth muscle actin as well as staining for alkaline phosphatase were performed after 10 and 14 days. Messenger ribonucleic acid expression of endothelial cell markers was highly upregulated in both basal and osteoblastic conditions after 5 days of coculture, indicating an endothelial cell differentiation, which was supported by immunocytochemistry for vascular endothelial growth factor receptor-1. Stromal derived factor-1 and vascular endothelial growth factor were highly expressed in MSC-MNC coculture in basal medium but not in osteoblastic medium. On the contrary, the expression levels of bone morphogenetic protein-2 and angiopoietin-1 were significantly higher in osteoblastic medium. Pericyte markers were highly expressed in both cocultures after 5 days. In conclusion, it was demonstrated endothelial cell and pericyte differentiation in MSC-MNC cocultures both in basal and osteoblastic medium indicating a potential for neovascularization for tissue engineering applications

Evidence for the in vivo existence and mobilisation of myeloid angiogenic cells and pericyte-like cells in wound patients after skin grafting

Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+CD45+CD31+CD34− MACs and CD14+CD45+NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRβ) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1−1.7-fold in all patients 1 day after skin grafting (p < 0.01). In addition, histological analysis demonstrated effective vascularization of skin grafts, as well as presence of pericytes, and CD14 and CD45 expressing myeloid cells during wound healing. In conclusion, our data shows, for the first time, the presence and mobilisation of MACs and pericyte-like cells in human circulation.publishedVersionPeer reviewe

Evidence for the in vivo existence and mobilization of myeloid angiogenic cells and pericyte-like cells in wound patients after skin grafting

Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+ CD45+ CD31+ CD34- MACs and CD14+ CD45+ NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRβ) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1-1.7-fold in all patients 1 day after skin grafting (p </p

Cilia-related protein SPEF2 regulates osteoblast differentiation

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