WO2023052820A1

WO2023052820A1 - Expression analysis of a specific gene pool to determine whether the population of adipose tissue-derived mesenchymal cells (at-msc) selected for clinical application can undergo a transformation into neoplastic cells

Info

Publication number: WO2023052820A1
Application number: PCT/IB2021/058990
Authority: WO
Inventors: Tomasz OŁDAK; Tomasz KOLANOWSKI; Igor STEPANIEC; Krzysztof MACHAJ
Original assignee: Polski Bank Komórek Macierzystych S.A.
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2023-04-06

Abstract

The object of the invention is a set of genes comprising at least one gene panel selected from: CDKN2A, CDH20, HAND2, PDGFR-a; or ALOX15, CDH9, DRD4, ESM1, HEY1, NKX2-5; or FUT3, PROM1, COL2A1, FOXA1, MY03B; or CLDN1, CPLX2, EOMES, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1; or TDGF1, DNMT3B, IDO1, NANOG, POUF5F1, SOX2; for use in the determination of the teratogenic potential of an AT-MSC mesenchymal cell population.

Description

Expression analysis of a specific gene pool to determine whether the population of adipose tissue-derived mesenchymal cells (AT-MSC) selected for clinical application can undergo a transformation into neoplastic cells

The invention relates to the use of the expression analysis of selected genes in adipose tissue- derived mesenchymal cells (AT-MSC) that will enable the determination of teratogenic potential of those cells. This will enable the selection of such cells that will not pose a threat of neoplastic transformation in therapeutic applications. More specifically, the invention relates to the use of the expression analysis of a specific gene pool to determine whether the AT-MSC population selected for clinical application can undergo a transformation into neoplastic cells. The invention will enable the selection of cellular preparations, wherein the safety of their use in patients is increased to the maximum.

Mesenchymal stromal/ stem cells (MSC) are cells preserving the nature of multipotent cells, found in many tissues of adult organisms. The guidelines of the International Society for Cellular Therapy describe in detail the nomenclature and cell classification criteria for mesenchymal pools (EM Horwit, K Le Blanc, M Dominici at al., 2005, Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position Statement, Cytotherapy 7, (5) 393-395.; M Dominici, K Le Blanc, I Mueller at al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, 2006, Cytotherapy 8 (4) 315-317). Although these cells are present in the skin, dura mater, synovial fluid, bone marrow or adipose tissue, only the last two tissues are their potential source for clinical use in relation to autologous therapy in adults. It is well known that it is possible to obtain MSC from prenatal tissues (umbilical cord, placenta, fetal membranes), but their acquisition is limited to a very short perinatal period and for this reason does not fulfil the needs of all patients requiring this type of therapy. On the other hand, cells that appear to be the most attractive from a scientific point of view, such as embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), remain, apart from legal and ethical issues, at the stage of basic research. The adipose tissue as a source of MSC, in comparison to the bone marrow, shows several significant benefits. First of all, it can be obtained in a non-invasive manner, which is more justified from the ethical point of view, and allows to obtain a larger number of cells, but above all, these cells retain a stable phenotype and significantly greater proliferation potential in a culture. In addition, AT-MSCs are able to differentiate into cells derived from all three germ layers (mesoderm, ectoderm and endoderm), including adipocytes, chondrocytes, osteocytes, neural cells and hepatocytes, rendering them universal in terms of the scope of application and thus making them cells of choice for autologous applications for individuals who do not have fetal cells deposited in the Banks (e.g., isolated from Wharton's jelly).

The ability of MSC to self-renew and differentiate into numerous cell lines in the in vitro system as well as the broad spectrum of growth factors produced by them gives the basis for treating those cells as potential medicinal products in the broadly understood cell therapy in both classical medicine and cosmetology. An empirical confirmation of this reasoning is the market presence of commercially available MSC preparations, such as: cartistem (allogeneic MSC cells registered in the EU for the treatment of osteoarthritis), prochymal (allogeneic MSC cells from marrow registered in Japan, New Zealand and Canada for the treatment of GvHD), darvadstrocel (allogeneic adipose-derived cells registered, among others, in the EU for the treatment of perianal fistulae). The role they play in the study of the therapeutic use of MSC derived from adipose tissue is evident from the fact that currently, the access data available on clinicaltrials.gov inform about 256 clinical trials using ASC or adipose stem cells or AT -MSC. The application panel of these cells covers virtually every branch of medicine, such as orthopaedics, neurology, surgery, internal diseases or the treatment of Cov-19 using AT-MSC. Preparation of the AT-MSC cell preparation in accordance with the adopted criteria is very time-consuming and imprecise. The criterion of cell morphology and their adhesion to the medium results in a fairly large freedom of interpretation of the analyzed image. The same applies to the ability of these cells to differentiate into three basic adipo-, chondro- and osteogenic lines; apart from the possibility of subjective evaluation of the tested preparation, the available tests give only a qualitative result without leaving the possibility of deducing the number or degree of differentiation affinity. The cell culture itself, depending on the medium and conditions used, may affect the final test result. It is also not indifferent how cells are passaged in this type of research; any detachment from the medium creates a lot of stress for the cells, which may later be reflected in their final image. An additional disadvantage of these tests is that they are time-consuming and last at least three weeks. Even though the assessment of cell phenotype is the most precise method of their characterization, it leaves a margin of subjectivity in the assessment of individual readings associated with, for example, a different way of gating cells during their cytometric analysis, depending on the researcher. Most importantly, however, none of these analyses includes even an approximate risk assessment of neoplastic transformation of the cell pool prepared for application. Although the reports about the potential oncogenic risk of AT-MSC and MSC are not fully confirmed and the research of this problem seems to be burdened with a large methodological error, even the occurrence of the problem could make some clinicians wary of their application.

Methods for assessing the purity of mesenchymal stem cell preparations are known in prior art. EP 3091084 discloses an in vitro method for evaluating and/or monitoring the purity of a cell preparation containing mesenchymal stem cells (MSCs), said method comprising measuring the level of expression of at least one growth factor expressed by this cell preparation, wherein the at least one growth factor is SDF-la and/or VEGF. The method of the invention further comprises comparing the measured expression level with a reference expression level. Mesenchymal stem cells of the cell preparation are isolated from tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta or peripheral blood. In a specific embodiment, mesenchymal stem cells are adipose-derived stem cells (ASCs). The level of expression of at least one growth factor expressed by said cell preparation according to the invention is assessed at the protein level, preferably by detecting and/or quantifying at least one growth factor detected in the cell culture supernatant. The level of expression is assessed at the RNA level, preferably using RT-PCR, RT-qPCR, Northern Blot and/or hybridization techniques. In US 20060166214, a CDKN2A marker for distinguishing mesenchymal stem cells from fibroblasts and a method for detecting and distinguishing mesenchymal stem cells using this marker due to its different level of expression in mesenchymal stem cells is disclosed.

The present invention provides an analysis of the teratogenic potential of each AT-MSC intended for clinical application, thus filling a gap in stem cell research and enhancing the safety of their use (or rather reducing concerns over its administration). The determination of an appropriate pool of genes recommended for testing in the present invention is a completely new quality in the evaluation of the broad clinical potential of prepared AT-MSCs. The proposed evaluation of cells using a real-time PCR technique increases the probability of detecting changes in the expression of selected genes that may indicate progressive deregulation leading to neoplastic transformation and eliminates the risk of previously described mistakes in the assessment of results caused by methodological factors. The study of the expression of genes encoding intranuclear (transcription factors) as well as cytoplasmic (structural and effector) proteins within the framework of proposed molecular tests allows to obtain results ahead of time, because the deregulation of the expression of transcription factors can inform in advance about the dysfunction of metabolism or the dysfunction of cell cycle regulation - namely, the information that is much more difficult to obtain based on the analysis of cellular material in a flow cytometer. In addition, expression tests are characterized by a much higher accuracy of the results (several hundred times) due to selected genes, among others, chosen because of their qualitative (negative or positive selection) or significantly different quantitative differences in expression. Additionally, in molecular tests, there is no factor of dependence on the used fluorochromes coupled to the same antibodies, introducing additional disturbances and decrease in the sensitivity of cytometric tests. It is also important that in the case of the analysis of the currently used surface markers, only selected proteins of the cell membrane are tested, i.e., the approximate resultant mechanisms occurring inside the cell. In the case of molecular analyses, any gene, often a transcription factor functioning at the beginning of a signal pathway, is tested, hence the study is even more biologically sensitive and enables faster analysis before its effects occur (if at all) on the cell membrane. In summary, RT-PCR provides repeatable and stable data due to the use of references and results in the form of relative expression of the studied genes. The proposed panel of genes, the expression of which is the object of the invention, represents a breakthrough in the obligatory process of quality control of mesenchymal cells of adipose origin intended for clinical use. The use of the proposed technique, on the one hand, enables the identification of AT-MSC without the need to use long-term cell cultures and on the other hand, enables the identification of the presence of cells with a potential carcinogenic risk in their population and the elimination of such batch of cells. At the same time, the use of molecular techniques allows to obtain much more sensitive, more accurate results with a smaller measurement error.

The protocol developed by the authors can be successfully used to differentiate the expression profile at the molecular level of MSC lines of adipose origin (both due to positive and negative selection) from human pluripotent cells (hIPSC) or selected neoplastic lines derived from 3 different germ layers. It is therefore a source of data that extends and complements the previously used AT-MSC quality control protocols, which enables the introduction of a new safety level in their use. The object of the invention is a set of genes comprising at least one gene panel selected from: CDKN2A, CDH20, HAND2, PDGFR-a; or ALOX15, CDH9, DRD4, ESMI, HEY1, NKX2-5; or FUT3, PROMI, COL2A1, FOXA1, MY03B; or CLDN1, CPLX2, EOMES, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1; or TDGF1, DNMT3B, IDO1, NANOG, POUF5F1, SOX2; for use in the determination of pluripotent and teratogenic potential of a mesenchymal cell population derived from adipose tissue (AT-MSC).

Preferably, mesenchymal cells are derived from adipose tissue obtained via liposuction or from fat fragments obtained from post-surgical waste material.

Preferably, the CDKN2A, CDH20, HAND2, PDGFR-a genes are a subset of genes indicating the characteristic expression profile of a mesenchymal cell population.

Preferably, the ALOX15, CDH9, DRD4, ESMI, HEY1, NKX2-5 genes are a subset of genes indicating the lack of teratogenic potential for mesodermal line neoplasms of a MSC population.

Preferably, the FUT3, PR0M1, COL2A1, FOXA1, MY03B genes are a subset of genes indicating the lack of teratogenic potential for ectodermal line neoplasms of a MSC population.

Preferably, the CLDN1, CPLX2, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1 genes are a subset of genes indicating the lack of teratogenic potential for endodermal line neoplasms of a MSC population.

Preferably, the TDGF1, DNMT3B, IDO1, NANOG, POUF5F1, SOX2 genes are a subset of genes indicating the lack of teratogenic potential for neoplasms of other origin of a MSC population.

The Applicant is the first to present the possibility of performing a thorough analysis of adipose tissue-derived MSC cells, which allows to clearly distinguish those cells from other lines with the ability to pluripotency, neoplasm formation as well as those derived from different germ layers.

Gene expression should be calculated from the base-2 logarithm of the relative expression for mesenchymal cells versus the mean expression for the control population of cells with adequate characteristics (mesenchymal cells, neoplastic mesodermal cells, neoplastic ectodermal cells, neoplastic endodermal cells, or pluripotent cells) that constitute the control population for a given panel, normalized for the reference genes: GAPDH, HPRT, and ACTB as well with the confidence interval (CI) = 95%.

Materials and methods:

Procedure 1 — Cell detachment and preparation for further procedures

Apparatus and materials:

• Laminar flow cabinet

• Inverted microscope

• Incubator

• Centrifuge • Culture dish with AT-MSC

• 3 ml Pasteur pipettes from Medlab

• Sterile 15/50ml Falcon tubes from Coming

• Klercide 70/30 from Ecolab

• Sterilesorb wipes from Contec

Reagents:

• MSC NutriStem complete culture medium from Biological Industries

• Human albumin from CSL Behring GmbH

• Tryple Select (IX) from Life technologies

• Sterile physiological saline solution from Fresenius Kabi

• Antibiotic-Antimycotic solution from Gibco

Detailed description of the procedure:

1. 4 days after the last passage (p4) of AT-MSC cells (if confluence was approximately 80%) in an incubator - 37°C, 5% CO2 in a complete culture medium - NutriStem™, remove the supernatant from the culture dish.

2. Wash the culture dish with a physiological saline solution.

3. Pour 1 ml/75 cm² of Tryple enzyme solution (IX) into the culture dish.

4. Close the culture dish, stir gently to allow the fluid level to equalize on each layer and place in an incubator for 5 minutes (37°C, 5% CO2).

5. After this time, check under the inverted microscope whether the cells changed to a spherical shape and detached from the surface.

6. If the cells are not detached, reinsert the culture dishes into the incubator and check after another 5 minutes.

7. Check the culture dish with the cells under the microscope again.

8. If the cells have detached, place the culture dish in a laminar flow cabinet.

9. Block the enzyme by adding a 5% albumin solution to the culture dish at 5: 1 ratio to the added volume of Tryple (IX).

10. Pour the contents of the dish into sterile plastic Falcon flasks.

11. Wash the culture dish with a physiological saline solution to rinse the bottom of the flask and then collect the remaining cells.

12. Pour the contents of the culture dish back into sterile Falcon tube.

13. Centrifuge the suspended cells in a centrifuge (7 minutes, 300 RCF, RT).

14. Count the centrifuged cells on the ADAM MC 2.0 device and estimate the viability.

15. Resuspend in a culture medium in a volume dependent on further procedures. Procedure 2 — Isolation of total RNA from mesenchymal stem cells (AT-MSC); Purification, concentration measurement and RNA quality control

Apparatus and materials:

• Single channel pipetes

• Centrifuge

• Thermomixer C

• Vortex

• Sterile RNase-free tubes

• Sterile tips with filters

• RNase ZAP (Ambion, AM9780)

• Sterile 20G (0.9 mm) RNase-free needles

• Sterile RNase-free syringes

Reagents:

• RNase-free water

• RNA isolation kit: Qiagen RNeasy Plus Mini Kit (Qiagen, #74104)

• Lyophilized DNase I with Reconstitution Buffer (1500 U) (Qiagen, #79254)

• 2M of dithiothreitol (DTT)

• 70% ethanol (molecular grade, RNase-free)

• 100% ethanol (molecular grade, RNase-free)

Detailed description of the procedure:

1. Centrifuge [5 minutes, RT, 1500 rpm] the cells (no more than 5xl0⁶) and remove the supernatant.

2. Add 350 pl of RLT buffer with DTT to the cell pellet.

3. To homogenize, mix the mixture thoroughly by vortexing or pipetting.

4. Cellular lysate should be subjected to additional homogenization by pipetting or by using a 20G needle and a RNase-free syringe (pipete the lysate at least 5 times).

5. Vortex for 1 min.

6. Add 350 pl of 70% ethanol and mix well by pipetting. Do not centrifuge.

7. Transfer 700 pl of the sample (together with the residue, if present) to the RNeasy column (RNeasy spin column from Qiagen RNeasy Plus Mini Kit) placed in a 2 ml collection tube.

8. Centrifuge for 30-60 sec at a speed > 8000 x g (> 10,000 rpm).

9. Remove the minicolumn, discard the flow-through and reinsert the minicolumn in the collection tube. 10. Add 350 pl of RW1 buffer to the column. Close and centrifuge for 30 sec at a speed of > 8000 x g (> 10,000 rpm).

11. Discard the flow-through according to step 10.

12. Add 80 pl of the mixture with DNase I directly to the column bed. Incubate at room temperature for 15 min.

13. Add 350 pl of RW1 buffer to the column (RNeasy spin column). Close and centrifuge for 30 sec at a speed of > 8000 x g (> 10,000 rpm).

14. Discard the flow-through according to step 10.

15. Add 500 pl of RPE buffer to the column (RNeasy spin column). Close and centrifuge for 30 sec at a speed of > 8000 x g (> 10,000 rpm).

16. Discard the flow-through according to step 10.

17. Add 500 pl of RPE buffer. Close and centrifuge for 2 min at a speed of > 8000 x g (> 10,000 rpm).

18. Transfer the column (RNeasy spin column) into a new 2 ml collection tube. Gently close and centrifuge at a maximum speed for 1 min.

19. Transfer the column (RNeasy spin column) to a 1,5 ml RNase free tube.

20. Add directly to the column bed of 30-50 pl of RNase-free water. Close and incubate for 10 min at room temperature.

21. Centrifuge for 1 min at a speed of > 8000 x g (> 10,000 rpm).

22. Analyse the flow-through further or freeze at -80°C.

Procedure 3 — Concentration measurement and qualitative assessment of total RNA

Apparatus and materials:

• Single channel pipette 0,5-10 pl

• UV cabinet

• UV-VIS BioDrop KAP61 spectrophotometer

• Vortex

• Sterile RNase-free tubes (0.2, 0.5 and 1.5 ml)

• Sterile RNase-free tips with filters, 10 pl

• RNase ZAP (Ambion, AM9780)

Reagents:

• RNase-free water

Detailed description of the procedure:

1. Thaw RNA samples and keep on ice. Transfer tubes with RNA to the BioDrop spectrophotometer. 2. Place the samples in the chamber. Turn on the spectrophotometer.

3. Vortex the RNA samples prior to concentration measurement.

4. Clean the BioDrop measurement port before measurement.

5. Calibrate the BioDrop against the reference sample (RNase-free water).

6. Perform the measurement using the set BioDrop parameters, i.e.:

•Pathlength: pLite 0.5 mm

•Dilution Factor: 1.000

•Background: On

•Units: ng/pl

•Factor: 50.00

7. Perform the measurements in duplicate for each sample .

8. RNA concentration is measured using a spectrophotometer at the wavelength of A260 nm. The value of the A260/A280 nm ratio should be 1.9-2.1; the value for A230/A260 nm should be above 1.8. These values indicate an appropriate degree of RNA purity.

Procedure 4 - Analysis of RNA quality and integrity on agarose gel

Apparatus and materials:

• Single-channel pipettes (0.5-10 ul, 2-20 ul, 20-200 ul, 100-1000 ul)

• Microcentrifuge with cooling function as well as suitable rotors and adapters

• Thermomixer C, thermoblock or dry bath

• Vortex

• Autoclave

• Microwave oven

• Sub-Cell agarose electrophoresis system (Mini or wide Mini) (Bio-rad)

• UV Transilluminator or Chemidoc XRS+ (Bio-rad)

• Sterile RNase-free tubes, 0.2 mL

• Sterile RNase-free tips with filters, 10 pL, 200 pL, 1000 pL

• RNase ZAP (Ambion, AM9780)

Reagents:

• RNase-free water (extremely pure)

• DEPC (solution)

• DEPC water

• Powdered agar (molecular grade) • TAE Buffer (Tris-acetate-EDTA) 50X (concentrated)

• Midori Green Advance (ABO)

• RiboRuler High range RNA ladder (#SM1821, Thermo Fisher Scientific)

• Loading dye for RNA 2X (95% formamide, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol FF, 0.025% ethidium bromide, 0.5 mM EDTA).

Detailed description of the procedure:

1. Weigh the required amount of molecular grade agar (depending on the gel size and concentration). To obtain the recommended 1,5 % gel, weigh 1.5 g of agar per 100 ml of TAE IX buffer.

2. Transfer the powdered agar to a sterile RNase-free glass bottle.

3. Depending on the number of samples, prepare 50-100 ml of TAE IX buffer in DEPC water (1 ml of concentrated TAE 50X buffer to 99 ml of DEPC water).

4. Mix powdered agar with buffer manually.

5. Dissolve the agar in the buffer using a microwave device (360 W power).

6. Cool the solution to approximately 60°C.

7. Add Midori Green Advance to dissolved agarose (5 pl/100 ml). Gently stir the solution, then pour the gel into the prepared casting tray with a comb.

8. Allow to stand for 30 to 60 minutes at room temperature to solidify.

9. Place Sub-Cell on ice. Level it. Fill with TAE IX buffer (in DEPC water).

10. After solidification, transfer the gel to Sub-Cell placed on ice.

11. Flood the gel with TAE IX buffer (2-5 mm above the upper line of the gel).

12. Thaw the High range RNA ladder and 2X loading dye on ice.

13. Mix the contents of the tubes by slowly pipetting or vortexing to ensure an even distribution of reagents within the vials.

14. Prepare the ladder from the following components: a. 2 pl of 2X loading dye, b. 2 pl of the RiboRuler High Range RNA ladder. c. 2 pl of RNase-free water

15. Vortex the sample and centrifuge rapidly.

16. Heat for 10 minutes at 70°C in a dry bath or thermoblock.

17. Immediately transfer to ice and cool for 3 minutes. Then, load the sample to the gel immediately.

Preparation and analysis of RNA samples:

1. Thaw the RNA samples on ice. 2. Mix the contents of the tubes by slow pipetting or vortexing to ensure an even distribution of reagents within the vials.

3. Combine the RNA samples (at least 100 ng) with the 2X loading dye in a ratio of 1 : 1 by volume.

4. Vortex the sample and centrifuge rapidly (at 4°C).

5. Heat for 10 minutes at 70°C in a dry bath or thermoblock.

6. Immediately transfer to ice and cool for 3 minutes. Then, load the sample immediately on the gel.

7. Load the RNA samples prepared in the previous steps and the RiboRuler High ladder Range RNA into the gel wells and run the electrophoresis for approximately 45 minutes until bromophenol contained in the loading dye (dark blue dye) migrates to the 2/3 of the gel. If the samples are not sufficiently separated, extend the electrophoresis for another 15 minutes.

8. Visualize samples using a UV transilluminator.

Procedure 5 - Analysis of DNA quality and integrity on agarose gel

Apparatus and materials:

• Single channel pipettes

• Cooling stand (4°C) for 0.2 ml test tubes

• Thermomixer C

• Vortex

• Autoclave

• Microwave oven

• Sub-Cell agarose gel electrophoresis system (Mini or wide Mini) (Bio-rad)

• UV Transilluminator or Chemidoc XRS+ (Bio-rad)

• Sterile DNase-free tubes, 0.2 ml

• Sterile DNase-free tips with filters, 10 pl, 200 pl, 1000 pl

• DNaseZAP (Ambion, AM9780)

Reagents:

• Nuclease-free water (exceptionally pure)

• Autoclaved water

• Powdered agar (molecular grade)

• 5 OX (concentrated) TAE buffer

• Midori Green Advance (ABO) • GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific)

• GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific)

• TriTrack 6X loading dye (10 mM Tris-HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 0.15% orange G, 60% glycerol and 60mM EDTA).

Detailed description of the procedure:

1. Perform steps 1-8 according to Procedure 1.

2. Place Sub-Cell on ice. Level it. Fill up to the line with TAE IX buffer (in autoclaved water).

3. After solidification, transfer the gel to Sub-Cell on ice.

4. Flood the gel with IX TAEbuffer (2-5 mm above the upper line of the gel).

5. Thaw the GeneRuler ladder and 6X TriTrack loading dye on ice.

6. Mix the contents of the tubes by slow pipetting or vortexing to ensure an even distribution of reagents within the vials.

7. Prepare the ladder from the following ingredients: a. 1 pl of 6X TriTrack loading dye, b. 1 pl of of GeneRuler ladder Ikb or 50 kb, respectively, c. 4 pl of nuclease-free water.

8. Vortex the sample and centrifuge rapidly.

9. Heat for 10 minutes at 70°C in a dry bath or thermoblock.

10. Immediately transfer to ice and cool for 3 minutes. Then, load the sample to the gel immediately.

Preparation and analysis of DNA samples:

1. Thaw DNA samples on ice.

2. Mix the contents of the tubes by slowly pipetting or vortexing to ensure an even distribution of reagents within the vials.

3. Combine DNA samples (at least 500 ng) with water and 6X TriTrack loading dye at a ratio of 1 :4: 1 by volume.

4. Vortex the sample and centrifuge rapidly (4°C).

5. Immediately transfer to ice and leave until the sample is loaded to the gel.

6. Load the DNA samples prepared in the previous steps and a suitable GeneRuler Ladder into the gel wells and run the electrophoresis for approx. 45 minutes until the orange G contained in the loading dye (orange dye) migrates to 2/3 of the gel. If the samples are not sufficiently separated, extend the electrophoresis for another 15 minutes.

7. Visualize samples using a UV transilluminator.

Procedure 6 - Reverse Transcription To perform a RT-PCR reaction, a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) along with its reagents was used. The reaction was carried out according to standard protocols for the C1000 thermocycler and in accordance with the recommendations of the RevertAid First Strand cDNA Synthesis Kit manufacturer. The obtained cDNA samples were protected by placing them in a freezer, -80oC.

Procedure 7 — Reaction of real-time PCR amplification (RT-PCR)

Reagents

• Nuclease-free water

• 2x POWERUP SYBR® Universal Master Mix buffer (Thermo Fisher Scientific)

• Forward primer 100 pM stock

• Reverse primer 100 pM stock

• cDNA template

To perform the qPCR reaction, the Thermal Cycler Cl 000 with the CFX-96 module was used. Primers were diluted at a ratio of 1 : 10 and 1 :25 in nuclease-free water. The reaction was carried out in three technical replicates per sample according to standard protocols for the thermocycler and POWERUP SYBR® manufacturer, which are known in the prior art, using the following reaction conditions: UDG activation [50°C for 2 minutes], polymerase activation [95°C for 2 minutes], 40 consecutive denaturation and elongation cycles [95°C for 15 seconds and 60°C for 1 minute]. Additionally, a step of checking the melting curve of tested genes was added for each tested sample.

Procedure 8 - Primer design

All PCR primers are designed according to standard procedures known in the art using NCBI primer BLAST, NetPrimer and NCBI Gene Base software.

Embodiments:

Example 1

Molecular Characteristics of Human Mesenchymal Stromal Cells, MSC

The cultured AT-MSC cell lines derived from adipose tissue (lipoaspirate) (n=4) were collected harvested at early and late passages (p3 and plO, respectively), maintaining the standards present in procedure 1, and were subjected to the RNA isolation procedure according to procedure 2. The resulting genetic material was measured for purity, quality and integrity according to the recommendations outlined in procedures 3 and 4. The isolated genetic material was used for comparative purposes with commercially available RNA of IPS pluripotency line, and six commercial (ATCC, Sigma Aldrich) neoplastic lines (ZR-75-30, A-375, HT-1080, A- 549, MCF-7, NCI-H727) of different germinal origin (researchers selected two lines per germ layer, previously tracing their origin based on detailed line descriptions provided by the manufacturer). After confirming the high quality of the obtained material, RNA was subjected to the reverse transcription procedure according to procedure 6. The single-strand cDNA thus obtained, completely free of genomic DNA (according to procedure 5), was used for real-time PCR amplification (according to procedure 7) using, among others, sets of primers designed on the basis of the information included in procedure 8 and calibrating them on the basis of standard curves. Based on extensive literature review in the field of cytology and oncology, a number of single and potentially specific markers for given populations were found and analysed to determine the nature of the cells cultured. Based on the cumulative data, 8 genes were finally selected (PR0M1, CDKN2A, FUT3, TDGF1, HER2, SOX9, B4GALNT1, TWIST1), which were used to differentiate between the populations, and it was also decided to extend the study with a set of genes included in the hPSC Scorecard™ (additional profile of 89 genes). When developing the RT-PCR-based experiment, two reference genes were always used (routinely GAPDH and HPRT, and in the case of predefined plates, GAPDH and ACTB). Reagent purity controls (- NTC), RNA controls, and a reference MSC assay for interplate calibration were loaded onto the plates. The collected data in the form of a multi-plate experiment were analyzed using commercial Maestro 1.1 software. The results of experiments in the form of genes selected from the collection were divided into six sets of neoplastic panels (MSC, pluripotency, non-specific neoplastic panels as well as panels characteristic of all three germ layers: endoderm, mesoderm and ectoderm) differentiating MSC populations from pluripotent lineages using the aforementioned predefined plates as well as self-designed primers.

In each panel, the obtained results of relative expression of individual genes (calculated using the "2-AACT" method approved by the scientific community and proposed in 2001 by the research group of Professor Livak in the article entitled:" Analysis of Relative Gene Expression Data Using RealTime Quantitative PCR and the 2-AACT Method") in relation to the control sample (depending on the panel, the AT -MSC line from the PBKM cell bank, the commercial IPS, ZR-75-30, A-375, HT-1080, MCF-7, A-549, and NCI-H727 lines, respectively), and the algorithm built into the Maestro 1.1 program was used to calibrate and eliminate potential differences in the reading between the individual plates. The final set of genes within each panel proposed by the panel makers is presented below:

• MSC panel (CDKN2A, CDH20, HAND2, PDGFR-a)

• Mesodermal panel (ALOX15, CDH9, DRD4, ESMI, HEY1, NKX2-5)

• Ectodermal panel (FUT3, PR0M1, COL2A1, FOXA1, MY03B)

• Endodermal panel (CLDN1, CPLX2, EOMES, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1)

• Pluripotency panel (TDGF1, DNMT3B, IDO1, NANOG, POUF5F1, SOX2)

Example 2 — Panel characteristic of the MSC cell population

In the panel developed by the inventors to differentiate MSCs from other lines, including the IPS pluripotent line and 6 neoplastic lines (two of lines characteristic of each germ layer), the average expression for the MSC biological group (n=4) was compared to the other lines. For this purpose, the values of relative expression of individual genes calculated using the approved "2-AACT" method in relation to the MSC line were analyzed. The inventors were looking for genes with expression, calculated on the basis of the MSC biological group with a 95% confidence interval, that does not overlap with the expressionin the other tested lines, and thus suggesting that they are potential characteristic markers of tested MSCs.. The screening of over 97 genes showed the presence of 4 such genes (CDKN2A, CDH20, HAND2, PDGFR-a). As developed by the inventors, the panel of genes characteristic of the AT-MSC population can be successfully used to differentiate the expression profile from human pluripotent cells (hIPSC), or neoplastic lines originating from 3 different germ layers, taking into account the confidence interval CI = 95%.

Example 3 — Panel characteristic of neoplastic lines of ectodermal cells

Subsequently, the inventors focused on the selection of gene groups that would enable an unambiguous differentiation of the AT-MSC cell line from the selected neoplastic lines at the molecular level.

Based on extensive subject literature screening, a list of genes regularly overexpressed in neoplastic cells was selected. Aware of the fact that so far, no characteristic neoplastic markers expressed in 100% of neoplastic cells have been found, the inventors decided to expand the selection panels by adding genes characteristic of individual germ layers (ectoderm, mesoderm and endoderm) from which selected neoplastic lines originated. It is obvious that MSC cells originated from mesoderm should not express any genes typical of lineages originating from other germ layers and the inventors unequivocally decided to prove that as well. Again, gene expression was calculated from the base-2 logarithm of the relative expression for mesenchymal stromal cells versus the average expression for the control population of ectodermal neoplastic cells that served as a control population (Fig. 2) for a given panel, normalized to reference genes: GAPDH, HPRT, and ACTB. The neoplastic markers selected by the inventors (FUT3 and PR0M1) differed in their expression to the highest extent from the tested AT-MSC samples and therefore they were included in this specific panel. However, each neoplastic line studied so far had a significantly increased expression of these genes relative to the AT-MSC biological group. As developed by the inventors, the panel of genes characteristic of neoplastic ectodermal lines can be successfully used to differentiate the expression profile of the AT-MSC line at the molecular level (due to negative selection) from human neoplastic lines originating from ectodermal germ layer, taking into account the confidence interval CI = 95%.

Example 4 — Panel characteristic of neoplastic lines of mesodermal cells

Another selection panel developed by the inventors enabled differentiation on the basis of the expression of genes characteristic for the neoplastic lineage of mesodermal origin, selected in relation to the MSC biological group. The markers (ALOX15, CDH9, DRD4, ESMI, HEY1, and NKX2-5) selected by the inventors differed in their expression to the highest extent from the tested MSC samples while remaining as genes characteristic of the mesoderm (a common lineage for both MSC cells and selected neoplastic lines originating from the mesodermal germ layer) and therefore they were included in this specific panel. Despite the convergence resulting from the common origin of the MSC samples, they did not express the ALOX15, CDH9 and NKX2-5 genes, in the case of the other tested genes the expression remained many times lower as compared to the average expression of control neoplastic lines. As developed by the inventors, the panel of genes characteristic of neoplastic ectodermal lines can be successfully used to differentiate the expression profile of the MSC line at the molecular level (due to negative selection) from human neoplastic lines originating from mesodermal germ layer, taking into account the confidence interval CI = 95%.

Example 5 — Panel characteristic of neoplastic lines of endodermal cells

Closing a group of selection panels related to genes characteristic of germ layers, the endodermal panel, enabled differentiating the populations based on the expression of genes characteristic for the neoplastic lineage of endodermal origin, selected in relation to the AT- MSC biological group. The markers (CLDN1, CPLX2, EOMES, FOXA2, HF1B, HNF4A, LEFTY1, and POU4F1) selected by the inventors differed in their expression to the highest extent from the tested MSC samples, while remaining as genes characteristic for the endoderm (characteristic of selected neoplastic lines originating from the endodermal germ layer) and therefore they were included in this specific panel.

A panel of genes developed by the inventors (Fig. 4), characteristic of neoplastic endodermal lines, can be successfully used to differentiate the expression profile of the MSC line at the molecular level (due to negative selection) from human neoplastic lines derived from endodermal germ layer, taking into account the confidence interval CI = 95%.

Example 6 — Panel characteristic of the pluripotent cell population

The last of the panels proposed by the inventors can be used successfully as an indicator of the pluripotency potential of studied cells. From all selected genes, six genes commonly considered to be strongly associated with the proliferation and maintenance of undifferentiated state of cells were finally selected. These include: TDGF1, DMNT3B, IDO1, NANOG, POU5F1, and SOX2. However, the purpose of the created panel was to prove that the studied AT -MSC populations do not exhibit pluripotent properties and thus can be safely used in therapies as a safe ATMP product. The developed panel of pluripotency showed that the tested lines of AT- MSC cells exhibit minimal expression (hundreds of times lower than in IPS cells) or no expression (as demonstrated for the TDGF1 and SOX2 genes) of selected pluripotency genes, taking into account the confidence interval CI = 95%.

Claims

1. A set of genes comprising at least one gene panel selected from: CDKN2A, CDH20, HAND2, PDGFR-a; or ALOX15, CDH9, DRD4, ESMI, HEY1, NKX2-5; or FUT3, PROMI, COL2A1, FOXA1, MYO3B; or CLDN1, CPLX2, EOMES, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1; or TDGF1, DNMT3B, IDO1, NANOG, POUF5F1, SOX2; for use in the determination of the teratogenic potential of a cell population.

2. The set of genes for use according to claim 1, wherein mesenchymal cells are derived from adipose tissue.

3. The set of genes for use according to claim 1, wherein the CDKN2A, CDH20, HAND2, PDGFR genes are a subset of genes indicating the characteristic expression profile of an AT- MSC cell population.

4. The set of genes for use according to claim 1, wherein the ALOX15, CDH9, DRD4, ESMI, HEY1, NKX2-5 genes are a subset of genes indicating the lack of teratogenic potential for mesodermal line neoplasms of the AT-MSC population.

5. The set of genes for use according to claim 1, wherein the FUT3, PR0M1, COL2A1, FOXA1, MY03B genes are a subset of genes indicating the lack of teratogenic potential for ectodermal line neoplasms of the AT-MSC population.

6. The set of genes for use according to claim 1 or 2, wherein the CLDN1, CPLX2, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1 genes are a subset of genes indicating the lack of teratogenic potential for endodermal line neoplasms of the AT-MSC population.

7. The set of genes for use according to claim 1 or 2, wherein the TDGF1, DNMT3B, IDO1, NANOG, POUF5F1, SOX2 genes are a subset of genes indicating the lack of teratogenic potential for neoplasms of other origin of the AT-MSC population.