Endothelial-derived Dll4 and PDGF-BB reprogram committed skeletal myoblasts to a pericytes fate without erasing their myogenic memory

Pericytes are vessel associated mural cells that form the smooth muscle layer of vessels. They are able to contribute to skeletal muscle regeneration as previously demonstrated for mesoangioblasts that indeed represent their in vitro counterpart. Pericytes are a heterogeneous population characterized by different marker like Alkaline Phosphatase, Smooth muscle actin, Neuro glial2 (NG2). Endothelial cells recruit pericytes from the surrounding mesoderm progenitor through a PDGF-PDGFrB loop. In this work I have conducted experiments whose results showed that functional pericytes may derive from direct reprogramming of already committed embryonic and, less frequently, fetal skeletal myoblasts. When co-transplanted in vivo with endothelial cells, Pax3 or Myf5 expressing myoblasts, downregulate myogenic genes, with the notable exception of Myf5, upregulate pericyte markers, adopting a perithelial position and morphology in newly formed vessel networks. The activity of endothelial cells can be replaced by exposure to PDGF-BB and Dll4 but not Dll1 or Jagged 1, while inhibition of Notch signaling via a γ-secretase inhibitor completely restores myogenesis, confirming that the skeletal myogenic program is not irreversibly erased. Notch activation in MyoD expressing embryonic cells in vivo abolishes myogenesis but not Myf5 expression that however cannot activate myogenin 7 and trigger myogenesis because Notch, beside suppressing MyoD transcription, activates Id and Twist factors that bind to and inhibit Myf5 transcriptional activity. Myf5 expressing cells activate pericyte genes and adopt a perithelial position, a phenomenon that can be rarely observed also in WT embryos. These data demonstrate that endothelial cells are able to directly reprogram committed skeletal myoblasts to mature pericytes for the formation and stabilization of vessel wall, suggesting that reprogramming occurs as a natural developmental process that leads in a sort of competition between endothelium and muscle

Crosstalk of Intercellular Signaling Pathways in the Generation of Midbrain Dopaminergic Neurons In Vivo and from Stem Cells

Dopamine-synthesizing neurons located in the mammalian ventral midbrain are at the center stage of biomedical research due to their involvement in severe human neuropsychiatric and neurodegenerative disorders, most prominently Parkinson’s Disease (PD). The induction of midbrain dopaminergic (mDA) neurons depends on two important signaling centers of the mammalian embryo: the ventral midline or floor plate (FP) of the neural tube, and the isthmic organizer (IsO) at the mid-/hindbrain boundary (MHB). Cells located within and close to the FP secrete sonic hedgehog (SHH), and members of the wingless-type MMTV integration site family (WNT1/5A), as well as bone morphogenetic protein (BMP) family. The IsO cells secrete WNT1 and the fibroblast growth factor 8 (FGF8). Accordingly, the FGF8, SHH, WNT, and BMP signaling pathways play crucial roles during the development of the mDA neurons in the mammalian embryo. Moreover, these morphogens are essential for the generation of stem cell-derived mDA neurons, which are critical for the modeling, drug screening, and cell replacement therapy of PD. This review summarizes our current knowledge about the functions and crosstalk of these signaling pathways in mammalian mDA neuron development in vivo and their applications in stem cell-based paradigms for the efficient derivation of these neurons in vitro

Conditional mutagenesis in the immune system: targeting the expression of the iCre2 recombinase to neutrophils and macrophages

Conditional mutagenesis allows the introduction of tissue specific mutations in the mouse and is of crucial importance in converting genome sequence information into functional data for biomedical research. Mice expressing the Cre recombinase in a spatially controlled manner are essential in creating such conditional knock-outs. A wide variety of Cre mice have been generated, but there is a distinct lack of models expressing the recombinase faithfully and at high levels in cells of the innate immune system. To address this need, three target genes, Itgb2l, Marco and Msr1, were chosen to create novel neutrophil and macrophage specific knock-in models harbouring iCre2, a recombinase engineered for increased expression levels. Two strategies were employed. Initially gene specific bacterial artificial chromosomes in which the iCre2 fragment replaced the endogenous translation start codon were created by Red/ET recombineering. Utilization of these BAC vectors for embryonic stem cell targeting successfully created knock-ins but the identification of homologous recombinants was complicated by the vectors’ large size. As the discovery of mutations impeding iCre2 functionality in the knock-in lines necessitated repeating the vector creation process, novel shorter vectors were designed. These vectors achieved targeting frequencies of around 10% and facilitated the isolation and verification of 9 Itgb2l and Marco specific iCre2 knock-in murine embryonic stem cell lines on the 129 genetic background. To determine tissue specific iCre2 expression before generating mouse models, an in vitro haematopoietic differentiation system, utilising three-dimensional embryoid body formation and selective expansion of progenitors in the presence of IL-3 and MCSF, was adapted. Embryonic stem cells were successfully differentiated into macrophages as assessed by CD11b and F4/80 marker expression. Collectively, this work has established the foundations for obtaining viable myeloid specific Cre producer mouse strains and discusses the potential of their future application in elucidating the role of macrophages and neutrophils in innate immune function

Bone marrow-derived cells in angiogenesis and cancer

Tumor is more than a mass of transformed cells. Tumor cells are embedded in the supporting tumor stroma, that is composed of cellular and non-cellular components. The cellular components include vascular cells, bone marrow-derived cells (BMDCs), and fibroblasts. In concert, tumor cells and stromal cells secrete proteases, cytokines, chemokines, and growth factors that modulate the tumor behavior. The mechanisms how stromal cells contribute to the tumor growth and tumor angiogenesis are still incompletely understood. This thesis aimed to study the role of BMDCs in angiogenesis and cancer. First, the role of BMDCs as a source of vascular endothelium was examined. The second study addressed the effects of an EGFR inhibitor, gefitinib, on the vascular cells and BMDCs in an EGFR-deficient B16 mouse melanoma tumor model. In the third study, chemokine-like prokineticins, that may have both angiogenic and immunomodulatory properties, were examined in a virus-associated human skin cancer type, Merkel cell carcinoma (MCC). BMDCs did not incorporate into the vascular endothelium in any of the experimental models, suggesting that the pre-existing vasculature is the main source of endothelium during angiogenesis. The potential of the BMDCs to differentiate into vascular endothelium may require specific molecular microenvironments, which needs further analysis. Treatment of B16 tumors with the EGFR inhibitor gefitinib reduced the pericyte number and coverage in the small CD31+ capillaries and the numbers of perivascular BMDCs suggesting that gefitinib treatment might have vascular and stromal effects in some tumors. The identity of the perivascular BMDCs that responded to treatment is not currently known, but the intimate perivascular location of the affected cells proposes that these cells might contribute to tumor angiogenesis via paracrine mechanisms. In MCC, higher than median tumor prokineticin-2 (PROK2) mRNA content was strongly associated with the presence of Merkel cell polyomavirus (MCPyV) DNA, viral large T antigen expression, higher than median number of tumor infiltrating CD68+ and CD163+ macrophages, and with favorable survival. The presence of prokineticin-1 (PROK1) mRNA in the tumor was associated with absence of MCPyV DNA and tended to be associated with poor survival. Neither PROK1 nor PROK2 mRNA content was associated with the tumor microvascular density. Taken together, these findings support the immunomodulatory role of prokineticins and suggest that prokineticins are involved in mediating the immune response in MCC, but their role in tumor angiogenesis in MCC requires further evaluation.Syöpäkasvaimet koostuvat syöpäsolujen lisäksi vaihtelevasta määrästä erilaisia strooman soluja, joita ovat mm. verisuonien endoteelisolut ja perisyytit, fibroblastit, sekä luuydinperäiset solut. Strooman solut erittävät proteaaseja, kemokiineja, ja kasvutekijöitä muodostaen soluväliaineen kanssa syövän mikroympäristön. Mikroympäristö voi estää tai edesauttaa syöpäkasvaimen kehittymistä. Mikroympäristön tutkiminen voi tuoda uutta tietoa syövän kasvun säätelystä ja voi auttaa syövän kliinisen taudinkulun ja uusiutumisriskin arvioinnissa. Väitöskirjatyön tavoitteena oli tutkia luuydinperäisten solujen merkitystä syövän kasvussa ja verisuonien muodostuksessa. Ensimmäisessä osatyössä selvitimme verisuonien endoteelin alkuperää hiirimallien avulla. On ehdotettu, että osa verisuonien endoteelistä olisi peräisin luuydinperäisistä progenitorisoluista. Tällaisen progenitorisolun olemassaolo mahdollistaisi uuden tavan estää tai lisätä verisuonien kasvua. Tulostemme perusteella luuydin ei näyttäisi olevan merkittävässä määrin verisuonien endoteelin lähde vaan verisuonien endoteelisolut ovat peräisin jo olemassaolevista paikallisista verisuonista. Toisessa osatyössä tutkimme epidermaalisen kasvutekjän reseptorin (EGFR) merkitystä verisuonien kasvun säätelyssä hiiren syöpämallissa, jossa syöpäsolut eivät ilmennä EGFR:ää. Tulostemme perusteella EGFR osallistuu verisuonien kasvun säätelyyn, sillä sen toiminnan estäjä gefitinib vähensi kasvaimen pienien verisuonien perisyyttien määrää sekä lähellä kasvaimen verisuonia sijaitsevien luudinperäisten solujen määrää. Kolmannessa osatyössä tutkimme prokinetisiinejä, kemokiinien kaltaisia peptidejä, ihmisen harvinaisessa ihosyövässä, Merkelin solukarsinoomassa. Tulostemme perusteella prokinetisiini-2:n korkeampi määrä kasvaimessa on yhteydessä kasvaimen Merkelin polyoomaviruspositiivisuuteen (MCPyV), CD68- ja CD163-positiivisten makrofagien suurempaan määrään sekä keskimääräistä parempaan eloonjäämisennusteen. Toisen ligandin, prokinetisiini 1:n, ilmentyminen kasvaimessa on yhteydessä kasvaimen MCPyV-negatiivisuuteen, ja huonompaan eloonjäämisennusteeseen. Kummankaan ligandin määrä kasvaimessa ei korreloinut kasvaimen verisuonien määrän kanssa, joten Merkelin solukarsinoomassa prokinetisiinit näyttäisivät osallistuvan ensisijaisesti kasvaimen immuunivasteen säätelyyn

Lineage Plasticity and Regenerative Potential of Adult Muscle Stem Cells: Investigation of Satellite Cell Direct-Reprogramming and Pericyte Self-Renewal

Satellite cells are responsible for most of adult skeletal muscle regeneration. Upon activation they differentiate into transient amplifying myoblasts that undergo cell fusion to form multinucleated fibres. Despite their remarkable differentiation ability and the positive outcomes obtained with transplantation in dystrophic mice and recently in patients with oculo-pharyngeal muscular dystrophy (OPMD), clinical trials in patients with Duchenne muscular dystrophy (DMD) showed limited efficacy, mainly ascribed to myoblasts low survival and poor migration ability. Muscle pericyte-derived mesoangioblasts (perivascular cells associated to the capillaries) also contribute to muscle regeneration and colonise the satellite cell niche. These cells can be injected systemically and migrate through the vascular endothelium, circumventing the necessity of multiple intra-muscular injections. Mesoangioblasts have been also tested in a recently completed phase I / II clinical trial to assess their safety profile in five DMD patients (EudraCT no. 2011-000176-33). We hypothesise that exploiting the key properties of myoblasts and mesoangioblasts may have the potential to produce clinically relevant cells, superior to those currently available. This work shows that exposure to molecules involved in pericyte specification such as the Notch ligand DLL4 and the growth factor PDGF-BB can induce direct reprogramming of primary satellite cells to pericyte-like cells. Reprogrammed cells acquire perivascular marker expression without losing the satellite cell marker Pax7. These highly myogenic cells can be expanded in culture and showed increased engraftment. In vitro and in vivo experiments also showed improved migration ability, similar to what has been observed with mesoangioblasts. Additionally, this thesis includes a set of experiments aiming to assess the self-renewal potential of mesoangioblast-derived cells via serial transplantation assays. Overall, the results obtained improve our understanding of smooth / skeletal fate choice and self-renewal, providing evidence of the possibility of exploiting a direct reprogramming approach to allow systemic delivery of myoblasts for cell therapies of muscular dystrophies

Aberrant transcriptional pathways in t(12;21) Acute Lymphoblastic Leukaemia

The single most frequent chromosomal translocation associated with childhood Acute Lymphoblastic Leukaemia is the t(12;21) rearrangement, that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TELAML1+ patients have a very good prognosis, relapses occur in up to 20% of cases and many patients face long-term side effects of chemotherapy. Our laboratory has previously shown that TEL-AML1 regulates Signal Transducer and Activator of Transcription 3 (STAT3) activation, which is critical for survival of the leukaemic cells. In this study, inhibition of STAT3 in TEL-AML1+ cells results in decreased SMAD7 gene expression. SMAD7 is an antagonist of TGF-β signalling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways. In order to investigate the role of SMAD7 in TEL-AML1+ leukaemia, lentiviral mediated SMAD7 knockdown was performed in human TELAML1+ cell lines. SMAD7 silencing inhibited proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. Furthermore, our data showed that this effect is not mediated through TGF-β signalling, indicating that SMAD7 was functioning through an alternative pathway. We also observed growth arrest following SMAD7 knockdown in other ALL and AML subtypes. Furthermore, silencing of SMAD7 in TEL-AML1+ ALL cells transplanted into immunodeficient mice impaired disease progression in vivo, resulting in prolonged disease latency. To investigate the essential pathways regulated by SMAD7 in these leukaemic cells, we performed RNA-sequencing analysis on TEL-AML1+ cells following SMAD7 knockdown. Global gene expression analysis revealed SMAD7 to be a regulator of cholesterol biosynthesis, a pathway critical for leukaemia cell survival. Our 4 experiments establish a novel transcriptional pathway operating specifically in t(21;21) ALL, but regulating downstream pathways essential for ALL in general. This study highlights new therapeutic opportunities for ALL

The Tumor Microenvironment of High Grade Serous Ovarian Cancer

The Special Issue on high grade serous ovarian cancer (HGSOC) and the contribution of the tumor microenviroment (TME) consists of reviews contributed by leaders in the OC field. As HGSOC metastases have a highly complex TME, there is an urgent need to better understand the TME in general, its distinct components in particular, and the role of the TME in the context of disease recurrence and development of chemoresistance. The Special Issue incorporates the current understanding of the different parts of thd TME components, including the cancer cells themselves, the cells surrounding the cancer cells or stromal cells, and the cells of the immune system, which are attracted to the site of metastases. In addition to these cells of the TME, the role of various cellular factors made by the cells of the TME are also the subject of the reviews. In addition, reviews in this Special Issue cover the complex relationships between the molecular mechanisms of HGSOC progression, including genomic, epigenomic and transcriptomic changes and changes in the immune cell landscape, as these may provide attractive new molecular targets for HGSOC therapy