Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57434—Specifically defined cancers of prostate
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers

Definitions

• the invention relates to a method of determining a gene combination that can identify in a very short time the potential of stem cells, typically human stem cells, of generating bone tissue.
• Muscoloskeletal diseases and disorders are more frequent than cardiac and respiratory diseases or tumors (see for instance :ip;// w i)oneandiointburden.orq/).
• stem cells As is known, there are various classes of stem cells, including: embryo stem cells, which are capable of generating any type of tissue, and adult stem cells, which are capable of generating tissues deriving from the same germ layer from which they are originated.
• MSC Mesenchymal stem/stromal cells
• MSCs must be capable of adhering to plastic, if they are maintained in standard culture conditions.
• CD1 1 b CD14, CD19, CD34, CD45, CD79a and class II HLA.
• MSCs must be capable of differentiating into osteoblasts, adipocites and chondrocites, under particular "ex vivo" stimulating conditions.
• BM bone marrow
• the first MSCs were isolated in 1974 from bone marrow (Friedenstein, 1 966). In bone tissue regeneration approaches therapeutic doses of about ten millions MSCs are infused per kilogram of patient weight.
• MSCs are from 0.001 % to 0.01 % the total cell populations.
• the amount of MSCs in the BM must be separated and enriched by cell culture (Dominici, 2001 ).
• GMP The production of cells required for clinical applications shall comply with existing good manufacturing practices, hereinafter briefly referred to as GMP.
• GMP standard have the purpose of ensuring that the cell product will be: sterile, pure, safe and functional.
• MSCs cultivated for clinical applications must be treated with animal derivative-free media.
• Production of cell therapies in GMP-accredited structures shall occur within a period of about three weeks, such that therapy administration will not be delayed and no changes will be introduced in the cell phenotype.
• Short cell isolation and growth times are critical for MSCs in autologous cell therapy, i.e. if the cells to be used have been obtained from the same patient to be later treated.
• MSCs of differentiating into bone "ex vivo" can be investigated by analyzing different stages of the process.
• Von Kossa staining highlights phosphate and carbonate anions deposited onto the matrix, whereas Alizarin staining reacts with calcium and other cations (Puchtler, 1 969).
• Both cytochemical methods can be used in the late stages of osteoblast maturation and have a reduced sensitivity.
• a prior art approach consisted in analyzing: alkaline phosphatase activity, the expression of type I procollagen and osteopontin in human MSCs implanted subcutaneously in a nude mouse model (Mendes, 2004).
• BSP bone sialoprotein
• OPN osteopontin
• OSX osterix
• the researchers analyzed the bone generation potential of MSCs by combining the use of a cytochemical test and molecular analysis.
• the first difference is based on the use of MSCs that are genetically modified with the TERT gene to become immortal.
• the second difference consists in that MSCs are treated with media containing animal derivatives.
• one object of the invention is to provide a potential assessment test that can define the quality of the cell product before administration to the patient and during cell expansion time.
• Another object of the invention is to improve the prediction of the bone tissue generation potential of MSCs by using a multi-parameter test that accounts for the various basic aspects for "in vivo" bone formation.
• These aspects comprise both the ability of expressing critical genes for bone formation and the ability to proliferate and deposit minerals into the matrix of the bone tissue.
• biomarkers which are part of the typical genetic complement of osteoblasts and whose expression levels are found to increase upon treatment of MSCs with an appropriate culture medium.
• MSCs from various BM donors were selected based on their ability to:
• This multi-parameter approach could mediate the heterogeneity of the different BM-MSC samples being tested.
• markers i.e. genes suitable for bone formation potential analysis.
• the “marker genes” have essential functions in the bone formation process, including:
• alkaline phosphatase i.e. coding an essential enzyme for bone matrix mineralization
• ⁇ coding protein components of the bone matrix i.e. Coding for producing collagen 1 A2 (COL1 A2), decorin (DCN) and elastin (ELN);
• the invention teaches how the osteogenic potential in a heterogeneous group of six BM-MSC donor can be defined from the analysis of five genetic markers.
• the invention relates to a method of determining a gene combination, as defined in the features of claim 1 .
• the invention achieves the following advantages: assessing the ability of MSCs to differentiate into bone, by analyzing the expression of five genetic markers, before administration to the patient.
• FIG. 1 a shows images of BM-MSCs induced into bone and stained by cytochemical staining with Alizarin
• FIG. 1 b shows microscope images of BM-MSCs induced into bone and stained by cytochemical staining with Von Kossa;
• FIG. 2 is a chart that represents the expression of the marker MKI67, at one week (white column) and at two weeks (grey column) respectively;
• FIG. 3 is a chart that shows the percents of formed bone (dark column), of biomaterial implanted in the animal model (grey column) and of other formed tissue (bright column) according to the individual donors and the control parameter CTL;
• FIG. 4 is a chart that represents the number of known and predicted interactions between the protein products of the five marker genes during the three bone differentiation stages;
• FIG. 5 is a chart that represents the expression of the five molecular biomarkers in BM-MSCs after one week's induction treatment
• FIG. 6a is a chart that represents the similarities in the expression of the five biomarkers in the six BM-MSC donors;
• FIG. 6b is a chart that represents the correlation coefficient "r 2 " between the expression of the biomolecular markets at one week and bone formation in the animal model of the six BM-MSC donors;
• FIG.7a is a chart that represents the expression of COL1 A2 in the donor #6 after treatment with the inhibitor SB431 542 and interferon-gamma (INF - Y);
• FIG. 7b are microscope images of the bone-induced donor #6, before and after treatment to decrease the expression of COL1 A2;
• FIG.8a is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of the five bio-markers at one week and the "in vivo" bone formation, using a COL1 A2 expression value for donor #6 defined as an average of the test values of the other donors capable of "in vivo" bone formation;
• FIG. 8b is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of the 5 bio-markers at two weeks and "in vivo" bone formation;
• FIG. 8c is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of: ALPL, COL1 A2, DCN and ELN, after one week's osteoinduction treatment, and "in vivo" bone formation;
• FIG. 8d is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of: ALPL, COL1 A2, DCN and RUNX2, after one week's osteoinduction treatment, and "in vivo" bone formation;
• FIG. 9a is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of: COL1 A2, DCN and RUNX2, after one week's osteoinduction treatment, and "in vivo" bone formation;
• FIG. 9b is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of: ALPL, COL1 A2, and RUNX2, after one week's osteoinduction treatment, and "in vivo" bone formation;
• FIG. 9c is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of RNX2 and DCN after one week's osteoinduction treatment and "in vivo" bone formation;
• FIG. 9d is a chart that represents the correlation coefficient "r 2 " between the similarity of the various donors in the expression of COL1 A2 and RUNX2 after one week's osteoinduction treatment and "in vivo" bone formation.
• Figures 1 a and 1 b represent the ability of BM-MSCs from six donors to deposit minerals into the matrix of the bone tissue.
• This method includes treatment in an appropriate culture medium consisting of the basal medium (nucleoside-free Alpha Medium added with 8% PL, 1 %
• Such basal medium was maintained for one week, and replaced every 2-3 days.
• BMP2 bone morphogenetic protein
• the MSCs were tested by cytochemical staining with Alizarin and Von Kossa.
• control sample A sample, herein referred to as "control sample” was treated with the basal culture medium only and was found in both cases negative to staining.
• BM-MSCs proliferative ability of BM-MSCs from six donors was tested by a molecular analysis on the gene MKI67. After one or two weeks' induction treatment, the BM-MSCs that exhibit a higher expression of such marker are those deriving from donors #1 , #3 and #4. The asterisks in the chart represent the statistic significance of these values as compared to those generated by the BM-MSCs of the same donor without no induction treatment.
• Figure 3 is a chart that shows the analysis of bone formation in an animal model.
• the dark grey column of the histogram represents the percent of bone in the overall area of the histological slide being tested.
• the symbol ⁇ represents the statistical significance of the percent of bone formed by each donor with respect to the control graft without BM-MSCs.
• BM-MSCs The mesenchymal stem/stromal cells of bone marrow, briefly BM-MSCs, from donors #1 , #2, #3 and #4 were found to be positive to Alizarin, whereas only BM-MSCs from donors #1 , #2 and #3 were found to be also positive to Von Kossa staining.
• the cells from donor #4 gave inconsistent functional results, i.e. were found to be: positive to Alizarin, negative to Von Kossa and negative to bone formation in the animal.
• the cells from donor # 6 also gave inconsistent results in terms of bone tissue generating ability, due to the lack of "ex vivo" mineralization and bone forming ability in the mouse.
• the cells from donor # 5 were found to be negative to both "ex vivo" and "in vivo” assays.
• the symbol * represents the statistical significance of the percent of bone formed by the various BM-MSC donors with respect to the BM-MSC graft from donor #5.
• the lack of a direct correlation between positive "ex vivo" mineralization assays and bone formation in the animal model involves the need to identify a more reliable bone differentiation potential assay.
• the chart shows that, after one week's induction treatment on the cells, heterogeneous gene expression levels are obtained.
• This method includes treatment with an appropriate culture medium consisting of the basal medium with: ⁇ -glycerophosphate, ascorbic acid, dexamethasone and bone morphogenetic protein (BMP2) for one week, with the medium being replaced every 2-3 days.
• an appropriate culture medium consisting of the basal medium with: ⁇ -glycerophosphate, ascorbic acid, dexamethasone and bone morphogenetic protein (BMP2) for one week, with the medium being replaced every 2-3 days.
• FIG. 6 there is shown a chart, known as dendrogram, in which the degree of similarity of gene expression in the five molecular markers from the 6 BM-MSC donors may be verified.
• the dendrogram establishes a hierarchy of similarities among the various donors, considering a single link and the euclidean distance.
• the donors #1 are #2 were found to be most similar, as well as the donor #3, and all are capable of "ex vivo” and "in vivo” bone formation.
• the analysis further associated the gene expression of donors #6 and #4, and particularly the donor #6 generated bone and the donor #4 did not generate bone "in vivo".
• the potential assay identified the difference between the donors #1 , #2, #3 and the BM-MSCs of the donor #6. More in detail, the donor #6 could not mineralize the matrix "ex vivo", but formed bone "in vivo".
• the dendrogram further shoes that the BM-MSC population from donor #5 is the most different from the others and was the only population that was negative to both "ex vivo" and "in vivo" functional tests.
• the BM-MSCs of donor #6 were treated, before osteogenic induction, with two molecules that can modulate collagen expression.
• the first molecule is interferon gamma (INF-Y), which is present in the human body, and the second molecule is a synthetic molecule named SB431 542 which modulates the TGFp signal, which in turn regulates collagen expression.
• IGF-Y interferon gamma
• SB431 542 synthetic molecule named SB431 542 which modulates the TGFp signal, which in turn regulates collagen expression.
• the BM-MSCs from donor #6 were osteogenically induced.
• the symbol * represents the statistical significance of the change of collagen 1 A2 expression in INF-Y- or SB431 542-treated cells as compared with untreated cells from the same donor #6.
• the INF-Y- or SB431 542-treated BM-MSCs from donor #6 were treated for two weeks with a bone differentiation-inducing medium and stained with Alizarin.
• the INF-Y- or SB431 542-treated BM-MSCs from donor #6 are stained with a color similar to that of donor #1 .
• the charts were obtained using the so-called “single link-Euclidean distance” method. More in detail, referring to Figure 8a, the x-axis of the Cartesian diagram represents the average percent values of "in vivo" bone formation, whereas the y-axis represents the correlation values of the expression of the four molecular markers from the six donors after standardization of the value of COL1 A2 expression in donor #6, using a middle value of the expression of the same gene in donors capable of forming bone "in vivo".
• the line obtained by joining the dots represented in the chart describes the progress of correlation between the molecular data of donors and bone formation in the animal.
• the slope of such line indicates the absolute value of the correlation coefficient, referenced to as "r 2 " whose maximum value coincides with 1 .
• the slope of this line has a value corresponding to 0.9482, and as a result the analysis of markers at one week describes a strong correlation between marker values and the percent of "in vivo" bone formation.
• the chart of Figure 8b comprises a Cartesian diagram which shows only the molecular data analyzed after two weeks' bone induction treatment: the chart shows a line whose slope is described by the value 0.5335.
• the results so obtained support the strategy of analyzing expression levels of biomolecular markers after one week's osteogenic induction.
• FIGS. 8c and 8d show Cartesian diagrams which analyze expression levels after selection of four biomolecular markers, analyzed after one week's osteogenic induction treatment.
• FIGS. 9a and 9b show respective Cartesian diagrams in which the expression levels are analyzed after selection of three biomolecular markers, after one week's osteogenic induction treatment.
• FIGS. 9c and 9d show Cartesian diagrams which analyze expression levels of two biomolecular markers, analyzed after one week's osteogenic induction treatment.
• the invention was conceived to use five biomolecular markers and analyze their results after one week's osteogenic treatment.
• the analysis can be based on more stringent parameters and is thus capable of producing more reliable results.
• the invention has been found to fulfill the intended objects.

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