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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
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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
  • C12N5/066—Tenocytes; Tendons, Ligaments
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
  • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
  • C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
• • 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
  • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor

Definitions

• This invention concerns a method for the in vitro production of tenocytes starting from mammal-origin mesenchymal stem cells. It also concerns a method for the characterisation of tenocytes, and a method for the characterisation of mammal-origin mesenchymal stem cells.
• This invention applies in general to the treatment of mammals affected by tendon injuries.
• leg tendons including the flexor digitorum superficialis and the flexor profundus tendons
• leg tendons including the flexor digitorum superficialis and the flexor profundus tendons
• a tendon mainly consists of fibrous connective tissue which is collagen-based (mainly type I, but also type III and V) , proteoglycans, elastin and fibronectin and, when torn, does not spontaneously regenerate.
• the base material of fibrous connective tissue is produced by tenocytes, highly specialised cells enclosed in the extracellular matrix they produce. Also because of their high degree of specialisation, tenocytes nevertheless present a very limited replication potential, so that attempts to obtain a long-lasting and efficient in vitro reproduction have proved unsatisfactory.
• the presence of tenocytes in a cell culture is typically identified by means of cytofluorimetric analyses, histochemical staining or immunological tests.
• MSC mesenchymal stem cells
• MSCs appropriately cultivated and enriched in vitro, directly in the injured tendon so that at least a part of these cells are induced to differentiate into tenocytes by factors produced by cells present in the surrounding microenvironment and can thus regenerate the tendon material .
• MSCs can be easily cultivated in vitro and demonstrate a good degree of proliferation.
• the need still remains, however, to develop new methods for the in vitro production of tenocytes, making it possible to develop effective protocols for the repair of tendon damage in mammals, in particular in racehorses.
• this sector there is also the need to establish effective protocols for the characterisation of MSCs and of tenocytes.
• the basic problem of this invention is the provision of a method for the in vitro production of tenocytes, in order to satisfy the requirement described above.
• FIG. 1 is a collection of photographic reproductions illustrating the results of a Reverse Transcriptase Polymerase Chain Reaction (hereinafter, in brief, RT-PCR) carried out to identify the presence or non-presence of the expression of certain genes within a cell culture of MSCs;
• RT-PCR Reverse Transcriptase Polymerase Chain Reaction
• FIG. 2 is a collection of photographic reproductions illustrating the results of a RT-PCR carried out to identify the presence or non-presence of the expression of certain genes within a cell culture of tenocytes obtained by MSC differentiation.
• a first aspect of the method for the in vitro production of tenocytes comprises the steps of obtaining a cell culture of mammal-origin mesenchymal stem cells and of placing them in direct contact with an effective quantity of bone morphogenetic protein 12, (hereinafter, in brief, BMP12) .
• BMP12 bone morphogenetic protein 12
• the MSCs can be obtained from adult mammals or from fetal tissues, and in particular it is preferable that they be taken - A - from bone marrow or umbilical cord.
• the MSCs must first be appropriately isolated from the other cells in the harvested tissues and then placed in a culture medium that is suitable for their rapid proliferation.
• the MSCs are identified as such by means of a characterisation method that constitutes the second aspect of this invention, and foresees RT-PCR analysis of a sample of cells in order to identify the presence in the cells of expression products of specific genes, in particular of one or more genes chosen from among SOX2, OCT-4 and NANOG.
• the expression products of the genes in question will also be identified in the test which follows as "markers”.
• the aforesaid genes express respective transcription factors considered essential for the maintenance of undifferentiated cells and the presence of the markers they express is considered a clear indication of stem cell properties. It should be noted, however, that the expression of OCT-4 and NANOG genes is a typical characteristic of embryonal type stem cells, but that, surprisingly, it has also been found to be present in MSCs harvested from adult tissues.
• the RT-PCR analysis includes identification of the presence of the expression product of the CD34 gene.
• CD34 expresses a glycoprotein typically present in bone marrow and umbilical cord but not in MSCs. Analysis of the presence of the expression product of this gene thus represents a negative control, and the absence of CD34 in the sample of analysed cells confirms that the isolation of the MSCs from the bone marrow or umbilical cord has been carried out correctly.
• the analysis is performed by means of RT-PCR, comprising a first step of transformation into complementary DNA (hereinafter, in brief, cDNA) of messenger RNA (hereinafter, in brief, mRNA) isolated from the cell sample by a subsequent step of amplification by means of Polymerase Chain Reaction (hereinafter, in brief, PCR) of the complementary DNA obtained in the first step.
• cDNA complementary DNA
• mRNA messenger RNA
• PCR Polymerase Chain Reaction
• the step of amplification by means of PCR is preferably carried out by using some of the pairs of triggers specified in table 1.
• the pair of triggers respectively sense and antisense, defined by the SEQ ID N0:l and SEQ ID NO : 2 nucleotide sequences, was used for the SOX2 gene;
• the pair of triggers respectively sense and antisense, defined by the SEQ ID NO : 3 and SEQ ID NO : 4 nucleotide sequences, was used for the OCT-4 gene;
• the pair of triggers, respectively sense and antisense, defined by the SEQ ID NO: 5 and SEQ ID NO: 6 nucleotide sequences was used for the NANOG gene; while the pair of triggers, respectively sense and antisense, defined by the SEQ ID NO: 7 and SEQ ID NO : 8 nucleotide sequences, was used for the CD34 gene.
• BMP12 transforming growth factors D
• the amount of BMP12 added to the cell culture to induce the differentiation of MSCs into tenocytes is between 30 and 70 ng/ml and, preferably, 50 ng/ml .
• the direct exposure of the MSCs to the BMP12 is maintained for a period of 14-21 days.
• the presence of tenocytes in the MSCs treated with BMP12 is shown by gene characterisation, analysing a sample of the treated cells by means of RT-PCR in order to identify the presence of expression products of specific genes.
• RT-PCR analysis is carried out to identify the presence of expression products of genes coding a protein chosen between tenomodulin and decorin. These proteins play an important role in the formation and maintenance of the connective tissue of tendons, while they are completely absent in mesenchymal-origin undifferentiated cells .
• RT-PCR analysis is preferably carried out to identify the presence of both these proteins, and is also carried out to ascertain if the cell sample exposed to BMP12 contains the expression product of the gene coding lipocalin P19.
• This protein is present in various mesenchymal-origin tissues, such as for example in uterine and endometrial tissue, but is not present in tendons. Its absence therefore confirms the high selectivity of the differentiation of the MSCs obtained with the method according to this invention.
• the step of amplification by PCR for characterisation of the tenocytes is preferably carried out using some of the pairs of triggers specified in table 1.
• the pair of triggers respectively sense and antisense, defined by the SEQ ID NO: 9 and SEQ ID NO: 10 nucleotide sequences, was used for the tenomodulin coding gene;
• the pair of triggers respectively sense and antisense, defined by the SEQ ID NO: 11 and SEQ ID NO: 12 nucleotide sequences, was used for the decorin coding gene;
• the pair of triggers respectively sense and antisense, defined by the SEQ ID NO: 13 and SEQ ID NO: 114 nucleotide sequences, was used for the lipocalin coding gene.
• RT-PCR was also set up to ascertain the presence of the expression product of the GAPDH (glyceraldehyde-3-phosphato dehydrogenase) coding gene.
• GAPDH glycosylase dehydrogenase
• This "marker” is certainly present in both types of cells examined, and its presence thus represents a positive control of the correctness of the RT-PCR analysis.
• the pair of triggers, respectively sense and antisense, used for the amplification step by PCR of the GAPDH gene is defined by the SEQ ID NO: 15 and SEQ ID NO: 16 sequences.
• the oligonucleotide sequences of the triggers identified in this description with SEQ ID N0:l up to SEQ ID NO: 16 are reported in table 1 at the end of the description and in the format according to the international standard defined by the WIPO
• FCS fetal calf serum
• penicillin 100 mg/1 of streptomycin
• the cells were controlled for 3-4 days, then the culture medium was replaced with fresh medium similar to the previous mixture but with the addition of 50 ng/ml of EGF (epidermal growth factor) . In the following two weeks, the cells were trypsinised, counted, seeded in 25 cm 2 culture flasks (4 x 10 5 cells per flask) or on 24-well plates (2 x 10 5 cells/ml) and incubated at 37°C with 5% of CO 2 .
• EGF epidermal growth factor
• RNA of a sample of cell culture was isolated by means of the RNeasy Kit (Qiagen) , resuspended in 30 Dl of water free of ribonuclease and maintained at a temperature of -80 0 C.
• 0.5 mg of RNA was then converted to cDNA by means of reverse transcriptase (RevertAid H minusM-MLV RT by Fermentas Int., USA) and approx. 2 Dl of the obtained cDNA were amplified in a IX PCR solution, using 5U of Taq Gold polymerase (Applied Biosystem, USA) and 0.5 DM of triggers (sense and antisense) able to pair at the ends of the involved gene segments.
• PCR analysis was set up to amplify the cDNA of the SOX2, OCT-4, NANOG, CD34 and GAPDH coding genes.
• the trigger pairs used for this purpose are respectively identified by the sequences:
• SEQ ID NO: 3 SEQ ID NO : 4 ;
• oligonucleotide sequences were obtained from alignment studies with known gene sequences of other mammals, including man and mouse.
• the PCR was carried out as follows: a first heating step at 95°C for 10 minutes was followed by 35 work cycles, each consisting of 45 seconds at 95°C, 30 seconds at 58°C, 30 seconds at 72°C and a final step of 5 minutes at 72°C.
• SOX2, OCT-4 and NANOG coding genes are present in the amplified cDNA, indicating that mRNA coded by these genes, and consequently the product of their gene expression, was present in the total cell RNA analysed. On the contrary, the CD34 coding gene is absent, meaning that this gene is silent.
• OCT-4 and NANOG coding genes reveals the presence of MSCs, while the absence of the CD34 "marker" confirms the mesenchymal nature of the original bone marrow cells. Differentiation into tenocytes
• BMP12 was subjected to RT-PCR analysis to check that the MSCs had differentiated into tenocytes.
• RNA of a cell culture sample was isolated by means of the RNeasy Kit (Qiagen) , resuspended in
• RNA was then converted to cDNA by means of reverse transcriptase (RevertAid H minusM-MLV RT da Fermentas Int., USA) and approx. 2 Dl of the obtained cDNA were amplified in a IX PCR solution, using 5U of Taq Gold polymerase (Applied Biosystem,
• the PCR amplification step was set up to demonstrate the presence of the tenomodulin, decorin, lipocalin
• the trigger pairs used for this purpose are respectively identified by the sequences:
• the PCR was carried out as follows: a first heating step at 95°C for 10 minutes was followed by 35 work cycles, each consisting of 45 seconds at 95°C, 30 seconds at 58°C, 30 seconds at 72°C and a final step of 5 minutes at 72°C.
• the presence in the cell culture of expression products of the tenomodulin and decorin coding genes indicates the presence of tenocytes, while the absence of the expression products of the lipocalin P19 coding gene confirms that the differentiation process induced by BMP12 on the MSCs did not give rise to cells other than tenocytes, thus proving to be highly specific and selective.
• SEQ ID NO: 2 R GGGCAGTGTGCCGTTAAT
• OCT-4 SEQ ID NO: 3 F TCCCAGGACATCAAAGCTGCAGA
• SEQ ID NO : 4 R GTCAAACTTACGTACCCTCTCGGGTCT
• NANOG SEQ ID NO: 5 F TACCTCAGCCTCCAGCAGAT
• SEQ ID NO : 6 R CATTGGTTTTTCTGCCACCT
• CD34 SEQ ID NO : 7 F ATTACACGGAAAACGGTGGA
• SEQ ID NO : 8 R AATTCGGTATCAGCCACCAC
• Tenomodulin SEQ ID NO : 9 F GATCTTCACTTCCCTACCAACG
• SEQ ID NO:10 R CTCATCCAGCATGGGGTC
• SEQ ID NO: 12 R TGAGATGCGAATGTATGAGAGA
• GADPH SEQ ID NO: 15 F CAACGAATTTGGCTACAGCA
• SEQ ID NO: 16 R CTGTGAGGAGGGGAGATTCA

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• 2008
  • 2008-07-17 IT ITVR2008A000079A patent/IT1394262B1/it active
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