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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0661—Smooth muscle cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/165—Vascular endothelial growth factor [VEGF]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
  • C12N2506/1323—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from skeletal muscle cells

Definitions

• the present invention relates to a method for obtaining in vitro a population of cells comprising essentially human smooth muscle cells (hCML) expressing calponin and SM-MHC from a sample of human muscle biopsy cells or from human muscle biopsy cells differentiated in vitro into skeletal muscle cells (hCMS).
• the invention also comprises a composition comprising isolated smooth muscle cells that can be obtained by such a method as a therapeutic composition intended for humans.
• the invention also relates to the use of these isolated smooth muscle cells for the preparation of a therapeutic composition for the replacement of CML.
• the invention relates to the use of these isolated smooth muscle cells for the treatment of ischemia, cancer or any disease requiring revascularization of damaged tissue.
• the subject of the present invention is the use of these isolated smooth muscle cells as vectors of active principle for the preparation of a therapeutic composition intended for humans requiring treatment with this active principle.
• SMCs Smooth muscle cells
• CMS skeletal muscle cells
• the subject of the present invention is a process for obtaining in vitro a population of cells essentially comprising human smooth muscle cells (hCML) expressing calponin and the heavy chain of smooth muscle myosin, called here SM-MHC ("SM-MHC" for "Smooth Muscle Myosin Heavy Chains”), from a sample of human muscle biopsy cells or from human muscle biopsy cells differentiated in vitro into skeletal muscle cells (hCMS )
• hCMS skeletal muscle cells
• human muscle biopsy cells not expressing CD31 and CD14, and, where appropriate, markers of B and T lymphocytes, and
• VEGF vascular endothelial growth factor
• human sequence VEGF vascular endothelial growth factor
• hCMS human smooth muscle cells
• the method according to the invention is characterized in that said hCMS do not express CD34 and CD14.
• the method according to the invention is characterized in that said hCMS do not express calponin and SM-MHC.
• the method according to the invention is characterized in that said hCML obtained in step A) express calponin and SM-MHC.
• the method according to the invention is characterized in that said hCML obtained in step A) do not express the MyoD gene.
• the method according to the invention is characterized in that said hCML obtained in step A) from human muscle biopsy cells differentiated in vitro into human skeletal muscle cells (hCMS), express CD56 and desmin in vitro. significantly less than the hCMS cells used in step A).
• hCMS human skeletal muscle cells
• the method according to the invention is characterized in that said hCML obtained in step A) express My5 and myogenin.
• the method according to the invention is characterized in that said culture medium used in step A) further comprises at least one growth factor, preferably of human sequence, chosen from the group of growth factors constituted by PDGF-BB (platelet-derived growth factor, BB homodimer, also referred to as bb homodimer), IGF1 (insulin-like growth factor type 1), FGFb (basic fibroblastic growth factor), HGF (growth factor hepatocytes) and TNF ⁇ (tumor necrosis factor alpha), TGF ⁇ and all other factors that may have a role in the proliferation or differentiation of SMCs.
• PDGF-BB platelet-derived growth factor, BB homodimer, also referred to as bb homodimer
• IGF1 insulin-like growth factor type 1
• FGFb basic fibroblastic growth factor
• HGF growth factor hepatocytes
• TNF ⁇ tumor necrosis factor alpha
• the method according to the invention is characterized in that said hCML are obtained in step A) from a sample of human muscle biopsy cells differentiated in vitro into skeletal muscle cells (hCMS), characterized in that that said hCMS are obtained from a sample of human muscle biopsy cells by a method comprising the following steps: a) slicing said muscle biopsy, b) e ⁇ zymatic dissociation of the fibers and muscle cells and separation of the individualized cells by filtration, c) culturing the cells of muscle origin thus obtained in an adherent cell culture reactor in the presence of a growth medium and / or differentiation followed, if necessary, by one or more expansion phases, d) identification of the cell types present at the different stages of the culture by the analysis of specific cell markers, e) choice of the culture stage during which the desired cell type is in a dominant proportion in the cell population, i) harvesting a cell population at the culture stage chosen in e), g) where appropriate, freezing the cells taken in step f), in particular at the
• step b) the culturing of the cells obtained in step b) on a culture plate in medium C until a degree of confluence of approximately 20 to 50% or until the appearance of the first myotubes, and then washing the cells in PBS buffer (phosphate buffered saline), in FCS (fetal calf serum) and then in medium C, culture in medium C on expanded or multi-stage tray units that can be carried out again to reach a degree about 90% confluence or the appearance of the first myotubes;
• PBS buffer phosphate buffered saline
• FCS fetal calf serum
• step f) the freezing of said cells thus obtained in step f) is carried out in medium
• Medium A - Medium MCDB 120 (Ham et al., 1988) modified: substitution of L-valine by D-valine, removal of phenol red and thymidine.
• Medium C - Medium B + FGFb (10 ng / ml) + dexamethasone at 1 ⁇ M.
• Solution D Saline Phosphate Buffer (PBS).
• the antibiotic used is gentamycin, especially at 50 ⁇ g per ml, or a mixture of penicillin and streptomycin (especially at 100 IU / ml and 100 ⁇ g / ml respectively).
• the method according to the invention is characterized in that said hCML are obtained from a sample of human muscle biopsy cells previously predifferentiated into hCMS obtained according to the method as described in the international application. patent published under the No. WO
• the culture stage during which the desired hCMS cell type is in significant proportion in the cell population is determined by the appearance of a cell population of CD56 + phenotype representing at least 50%, preferably at least 60%, 70%, 75% and 80% of the general population.
• said cell population of CD56 + phenotype representing at least 50%, preferably at least 60%, 70%, 75% and 80% of the general population also has at least one of the phenotypes, preferably minus 2, 3 and the 4 phenotypes, selected from the group of phenotypes consisting of CD 10+, CD 13+, desmin +, HLA class 1 and not expressing HLA class 2.
• the process for obtaining in vitro a population of cells essentially comprising hCML according to the invention and wherein said hCML process is obtained from a sample of human muscle biopsy cells differentiated in vitro. in vitro in skeletal muscle cells (hCMS), is characterized in that in step A), said culture medium comprising VEGF is MCDB 120 medium as described by Ham et al. (in vitro CeIl Dev Biol., 24, 833-844, 1998) and modified by substitution of L-Valine by D-Valine, removal of phenol red and thymidine.
• the method for obtaining in vitro a population of cells essentially comprising hCML according to the invention and in which process said hCML are obtained from a sample of human muscle biopsy cells is characterized in that in step A), said culture medium comprising VEGF is M199 medium (such as for example Medium 199 Gibco, Grand Island, NY).
• M199 medium such as for example Medium 199 Gibco, Grand Island, NY.
• the process for obtaining in vitro a population of cells essentially comprising hCML according to the invention is characterized in that in step A), said culture medium comprises 10 ng / ml. ml of VEGF.
• the method for obtaining in vitro a population of cells essentially comprising hCML according to the invention is characterized in that the human muscle biopsy from which said hCML are obtained directly or previously predifferentiated in hCMS, is a biopsy taken in any muscular territory, preferably in the muscular territory of the leg, of the individual, individual child or adult, from whom the sample is taken.
• the present invention comprises isolated human smooth muscle cells obtainable by the method of the invention, wherein said isolated human smooth muscle cells are characterized by expressing calponin and SM-MHC.
• the present invention provides a composition comprising isolated human smooth muscle cells obtainable or obtained directly from a sample of muscle biopsy cells. human or from human muscle biopsy cells differentiated in vitro into skeletal muscle cells by the method according to the invention, as a medicament.
• the present invention also includes the use of isolated human smooth muscle cells obtainable or obtained directly from a sample of human muscle biopsy cells or from human muscle biopsy cells differentiated in vitro into skeletal muscle cells. by the method according to the invention, or the use of the composition as a medicament according to the invention for the preparation of a therapeutic composition intended for humans, in particular intended for the individual from whom the biopsy cells originate muscle grown in step A) of said method.
• said therapeutic composition is intended for the replacement or transplantation of CML in humans, preferably the replacement or transplantation is of the autologous type.
• said therapeutic composition is intended for the prevention or treatment of cancers, preferably in prior or simultaneous administration to an anti-cancer treatment by chemotherapy or radiotherapy.
• perivascular cells pericytes or CML
• phenotypic and functional changes abnormal form, expression of new markers, weak association with EC, have a cytoplasmic extension that penetrates deep into the tumor parenchyma) in the tumor microenvironment (6-8), thus becoming a new target for anti-angiogenic therapies.
• a new therapeutic approach would be to normalize tumor vasculature before its destruction to facilitate drug delivery (for review see (9)).
• recent results demonstrate the effectiveness of tumor regression, using combination therapies, after stabilization and normalization of the tumor.
• tumor vasculature (10). This stabilization of the tumor vessels could be achieved by injecting the SMCs at the site of the tumor or at the periphery.
• This therapeutic approach (the injection of isolated human CMLs obtainable or obtained directly by the process according to the invention, with a view to normalization of the tumor vessels) will preferably be carried out only in association with chemotherapy or radiotherapy. This will require defining a "therapeutic window", during which the injection of SMCs will allow the effect of the largest anti-cancer treatments.
• the vascular "normalization" will ensure a more functional network, thus promoting the local distribution of drugs, their more homogeneous distribution and the oxygenation of the tumor necessary for the functioning of certain drugs. This will allow a faster and more extensive action of the drugs in the tumor, thus a decrease of the doses administered reducing a priori the severity and the frequency of the side effects. Finally, the rapidity and combination of actions will quickly limit the proliferation and therefore the phenomena of tumor resistance often observed.
• the cell therapy proposed here does not constitute a new form of treatment intended to replace the current treatments, but will intervene as a complement and / or potential synergy to the currently proposed chemotherapies and radiotherapies.
• said therapeutic composition is intended for the prevention or treatment of ischemia, in particular cardiac or lower limbs.
• the subject of the present invention is the use of isolated human smooth muscle cells obtainable or obtained directly from a sample of human muscle biopsy cells or from muscle biopsy cells. in vitro human differentiated in skeletal muscle cells by the method according to the invention, for the preparation of a composition for the "normalization" of tumor vasculature or post-ischemic revascularization.
• these cells could also be used as a medicament intended for therapeutic use for: atherosclerosis, chronic venous diseases, vascular malformations (such as angiomas).
• the present invention also relates to the use of isolated human smooth muscle cells obtainable or obtained directly from a sample of human muscle biopsy cells or from human muscle biopsy cells. differentiated in vitro into skeletal muscle cells by the method according to the invention, for the preparation of a composition intended for the prevention or treatment of atherosclerosis, artheritis, chronic venous diseases or vascular malformations, in particular angiomas .
• these cells can be used as a shuttle or vector in order to deliver therapeutic active ingredients such as drugs or anti-angiogenic or pro-angiogenic factors.
• the present invention also relates to the use of isolated human smooth muscle cells obtainable or obtained directly from a sample of human muscle biopsy cells or from human muscle biopsies differentiated in vitro into skeletal muscle cells by the method according to the invention, as a medicament, especially as a vector for the administration of an active ingredient or a therapeutic compound, characterized in that: said human smooth muscle cells isolated are transformed so as to express said active ingredient or therapeutic compound; or - said isolated human smooth muscle cells have been modified to contain said active principle or therapeutic compound that it is desired to administer. Also included in the present invention is the use of isolated human smooth muscle cells obtainable or obtained directly from a sample of human muscle biopsy cells or from human muscle biopsy cells differentiated in vitro into cells.
• skeletal muscle by the method according to the invention, said cells being capable of expressing an active principle or therapeutic compound or containing an active ingredient or therapeutic compound, for the preparation of a therapeutic composition intended for the prevention or treatment of diseases requiring treatment with said active ingredient or therapeutic compound.
• a therapeutic composition intended for the prevention or treatment of diseases requiring treatment with said active ingredient or therapeutic compound.
• the use of isolated human smooth muscle cells obtainable or obtained directly from a sample of human muscle biopsy cells or from human muscle biopsy cells differentiated in vitro into skeletal muscle cells by the method according to the invention, for the preparation of a therapeutic composition is characterized in that said composition is administered intravenously or by transplantation.
• Figures IA to IC Characterization of skeletal muscle cells cultured in a medium containing FGFb.
• Figure IA The flow cytometry analysis demonstrates that these cells express CD56, Desmin and CD90 but do not express CD31, CD14 and CD45. In each histogram, the black line corresponds to the cells labeled with a negative control antibody. The dotted line corresponds to the cells labeled with the marker-specific antibody indicated in each histogram. These histograms are representative of 6 samples.
• Figure 1B RT-PCR analysis.
• Figure IC Characterization of skeletal muscle cells cultured by immunocytochemical analysis.
• FIG. 2A Morphology of Skeletal Muscle Cells (CMS) in Medium Containing FGFb or VEGF.
• Figure 2B RT-PCR analysis of skeletal and smooth muscle cell-specific gene expression in CMS grown in FGFb- or VEGF-containing medium. The cultures were harvested to prepare FARN at the different times indicated. RT-PCR was performed and PCR products were analyzed on agarose gels containing ethidium bromide.
• FIG. 3A Photos taken with a phase contrast microscope. Endothelial cells (ECs) and muscle cells are deposited together on the surface of a collagen gel. After 24-48 hours, the ECs interact with the SMCs, resulting from the differentiation of the umbilical cord blood precursors (FIG. 3A), or the SMCs obtained after culturing the skeletal muscle cells (FIG. 3C), to form networks. In contrast, CMS can not form networks under these conditions ( Figure 3B).
• FIG. 4A Cups of matrigel, marking HES. Co-injection of ECs and SMCs obtained from skeletal muscle cells, leads to the formation, in the implant, of vascular lakes ( Figure 4B), with the presence of red blood cells (set of points at the level of the arrows, Figure 4C at higher magnification). On the other hand, under these same conditions, the CMS do not form a functional vascular network (FIG. 4A).
• Figures 5A to 5F Photos taken with a phase contrast microscope of CMS (FIGS. 5A to 5D) and of CML (FIGS. 5E and 5F) cultured in a medium containing 20% fetal calf serum (FCS) (FIGS.
• FCS fetal calf serum
• Endothelial progenitor cells obtained as described in (16) are used as a positive control for the expression of VEGF (VEGFR) and FRS negative control receptors.
• VEGFR VEGF
• FRS negative control receptors FRS negative control receptors.
• CMS and CML do not express VEGFRl.
• VEGF decreases VEGFR2 expression but stimulates expression of FRS mRNA.
• SMCs Ex vivo differentiated SMCs from the precursors contained in the cord blood were obtained as previously described (16). They were cultured on type I rat tail collagen (60 ⁇ g / ml, SIGMA) in Ml 99 medium (Gibco) supplemented with 20% 20% fetal calf serum (FCS), 25 mM Hepes buffer (Gibco) and an antibiotic and antifungal solution (Gibco) and recombinant hVEGF at 10 ng / ml (R & D Systems) at 37 ° C, and in an atmosphere containing 5% CO 2 . The culture medium is changed twice a week. CMS were grown as previously described (12). To induce the differentiation of the cells into myotubes, the culture medium of the 80-90% confluent cells was changed with medium supplemented with 2% FCS, 25 mM Hepes and an antibiotic and antifungal solution. (Gibco). immunocytochemistry
• the cells were cultured on plates ("chamber slides", Lab Techn, PoIy Labo, France) and fixed with a 90% cold acetone solution. Primary antibodies were used. A human anti- ⁇ SMA murine monoclonal antibody (1A4, DAKO) and a mouse monoclonal anti-human smooth muscle myosin heavy chain monoclonal antibody (SMMS-I, DAKO).
• SMMS-I mouse monoclonal anti-human smooth muscle myosin heavy chain monoclonal antibody
• the EnVision TM System Peroxidase (DAB) kit (DAKO) was used to reveal ⁇ SMA and SM-MHC. The cells were ultimately counter-colored with haematoxylin. Flow cytometry
• RNAXEL R reagent (EUROBIO, UKs, France) according to the supplier's instructions.
• the synthesis of the cDNA was carried out with the kit "lst strand cDNA synthesis kit for RT-PCR (AMV)" (Boerhinger Mannheim). Therefore, the cDNA fragment of interest could be amplified by PCR.
• the PCR mixture contained an IX reaction buffer, 1.5 mM MgCl 2 , 0.2 mM deoxynucleotide mixture, 0.5 Taq polymerase unit, and 0.2 ⁇ M sense and antisense primers.
• A) Cellular population of departure The method of the invention relates to a method for obtaining a population of cells of which a dominant cell type is the type of smooth muscle cells. This method can be applied either directly to the muscle biopsy cells, or after a first phase of differentiation of the cells of the CMS biopsy and amplification of these cells.
• the conditions for obtaining muscle biopsies and CMS from these biopsies as well as their phenotypic characterization are defined in the international patent application published under No. WO 01/94555 (J.P. Marolleau et al.).
• the cells of the biopsy do not express CD31 and CD14.
• CMS From a few grams of a muscle biopsy, it is possible to obtain several hundred million CMS. These cells express CD56, desmin and myogenesis genes such as Myf5 and myogenin. On the other hand, they do not express CD34, CD14, and markers specific for SMCs such as: calponin and SM-MHC. These cells are finally able to fuse and give birth to multinucleated myotubes.
• Medium E M199
• antibiotic penicillin, streptomycin and, if necessary, an antimycotic (such as fungizone 25 ⁇ g / ml, or as indicated above).
• Medium G Medium F (M199 + SVF + Hépes + antibiotic) + VEGF (10 ng / ml).
• Medium H Medium B (MCDB + SVF + antibiotic + dexamethasone) + VEGF (10 ng / ml).
• EXAMPLE 2 Differentiation of Skeletal Muscle Cells into Smooth Muscle Cells Muscle biopsy cells were first cultured for expansion in medium containing FGFb as previously described (12). To characterize the phenotype of these cells, flow cytometry (FACS), reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry analyzes were performed. FACS analysis showed that most of these cells are positive for CD56 (80.30 + 19.50%), desmin (92.30 + 8.48%) and CD90 (91.32 + 10.19). %) and negative for the CD31 endothelial marker, the CD14 monocyte marker, and the CD45 leukocyte marker (Figure 1A).
• RT-analysis PCR has demonstrated that the cells express markers linked to myogenic cells such as My £ 5, MyoD and Myogenin (Figure 1B).
• the cells also express markers specific for SM22 ⁇ smooth muscle cells (FIG. 1B) and ⁇ SMA (FIG. 1C). But some isoforms of smooth muscle cells have been detected in skeletal muscle cells developing or regenerating (14, 15).
• the cells do not express markers of differentiated smooth muscle cells such as calponin ( Figure 1B) and SM-MHC ( Figure 1C).
• the type I collagen gel culture (3D culture) (BD Biosciences, Bedfoed, MA) was carried out according to the supplier's recommendations, namely: 0.5 ml of type I to 1 rat tail collagen mg / ml (Becton Dickinson) is poured into culture dishes 35 mm in diameter (Nunc, Fisher Scientific, Elancourt, France) and allowed to polymerize for 1 hour at 37 ° C. 400,000 cells in total (200,000 of each of the cell types when mixing the endothelial cells with the muscle cells) are then deposited on the surface of the gel and cultured for 24 hours under the different culture conditions. The formation of vascular networks is then observed under a phase contrast microscope and with a Kappa CFl IDSP charge-coupled camera. Results (see FIGS. 3A to 3C):
• OJ A 0.2 ml implant of matrigel (BD Biosciences) (containing 0.5 mg / ml of FGFb) is injected subcutaneously on the back of the NOD-SCID immunosuppressed mice.
• mice In the morning, the mice are irradiated sub-lethally (325 rad). In the afternoon, 500,000 cells are injected intravenously via the tail vein.
• HES staining (hemalin, eosin, saffron) was performed and examined (magnification 4x, 40x).
• VEGF is involved in inducing the transition of the CMS phenotype to CML by increasing the expression of the serum response factor SRS (also called SRF for serum response factor)
• VEGF is a major regulator of blood vessel formation during organ development and in adults.
• the expression of VEGFR1 and VEGFR2 receptors was analyzed in CMS and SMC.
• RT-PCR analysis shows that CMS express a large amount of VEGFR2. But when these cells are cultured in medium containing VEGF a decrease in VEGFR2 expression is observed ( Figure 6). And, regardless of the culture conditions, we did not demonstrate the detection of VEGFR1 expression.
• Jain R.K Normalizing of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005; 307: 58-62.
• Frid MG, KaIe VA, Stenmark KR Mature vascular endothelium can be delivered to smooth muscle cells via endothelium-mesenchymal transdifferentiation. In vitro analysis.

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