WO2021191853A1

WO2021191853A1 - The use of a gene panel to determine the teratogenic potential of mesenchymal and perinatal tissue-derived cells

Info

Publication number: WO2021191853A1
Application number: PCT/IB2021/052521
Authority: WO
Inventors: Tomasz OŁDAK; Tomasz KOLANOWSKI; Igor STEPANIEC
Original assignee: Polski Bank Komórek Macierzystych S.A.
Priority date: 2020-03-27
Filing date: 2021-03-26
Publication date: 2021-09-30
Also published as: EP4127239A1; PL433374A1; PL241520B1

Abstract

The subject of the invention is a set of genes comprising at least one gene panel selected among: CDKN2A, CDH20, HAND2, PDGFR-α; or ALOX15, CDH9, DRD4, ESM1, HEY1, NKX2- 5; or FUT3, PROM1, 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 mesenchymal cell (MSC) population or a perinatal cell population.

Description

The use of a gene panel to determine the teratogenic potential of mesenchymal and perinatal tissue-derived cells

The invention pertains to the use of a gene panel to determine the teratogenic potential of mesenchymal and perinatal tissue-derived cells. More specifically, the invention pertains to the use of genes to determine the potential of mesenchymal and perinatal tissue-derived cells to transform into tumor cells in order to increase the safety of products based on mesenchymal and perinatal tissue-derived cells.

Mesenchymal cells (also called mesenchymal stem cells or mesenchymal stromal cells; MSCs) are multipotent, non-hematopoietic cells present in multiple tissues of the body, capable of self renewal and able to transform into various types of mature cells, including adipocytes, osteocytes, chondrocytes, myocytes or nerve cells. There are many sources from which MSCs can be isolated: bone marrow, adipose tissue, synovial fluid, dura mater, skin, as well as prenatal tissues, such as: Wharton’s jelly, umbilical cord blood or the placenta and the fetal membranes. Despite the extensive presence of these cells in organisms, mostly cells derived from fetal tissues (primarily Wharton’s jelly and umbilical cord blood), bone marrow and adipose tissue are currently used for potential clinical therapy applications for utility reasons. However, it has been discovered that the individual populations differ slightly due to the impact of their microenvironment of origin. In research and clinical studies, the mesenchymal stem cells used most frequently are those derived from bone marrow, adipose tissue and Wharton’s jelly. From the point of view of their application, Wharton’s jelly-derived MSCs seem to be the most preferable, as the quantity of cells harvested from Wharton’s jelly exceeds other stem cell reservoirs in an adult organism, while the procedure itself is completely non-invasive (the product is generally harvested from umbilical cord, which constitutes medical waste), and the stem cells fraction obtained is relatively homogenous. The use of umbilical cord-derived cells is also much more ethically acceptable as compared with, for example, embryonic stem cells. The most recent achievements in the field of cell-based therapies, tissue engineering and regenerative medicine describe MSCs as potential biological products precisely because of their self-renewal and differentiation capabilities. MSCs have well-documented immunomodulatory and regenerative properties, contributing to their high therapeutic potential. MSC are already widely employed in therapies. The following stem cell -based medicinal products are currently commercially available: Cartistem (allogeneic MSCs approved in the EU for the treatment of arthrosis), Prochymal (allogeneic bone marrow-derived MSCs approved in Japan, New Zealand and Canada for the treatment of GvHD), Darvadstrocel (allogeneic adipose tissue-derived cells approved in the EU for the treatment of perianal fistulae). Therapies based on hematopoietic stem cells in hematology, treatment of non-healing wounds and bums or limbal stem cell transplantation, which have been the standard of care in Poland since 1989, cannot be overlooked here as well. In addition, there are hundreds of ongoing clinical studies with the use of MSCs in experimental therapies (263 studies as per clinicaltrials.gov).

Due to the profound interest in their potential applications in multiple branches of medicine, MSCs are being studied extensively to evaluate their viability, efficacy and safety. The few, albeit widely commented reports concerning the potential teratogenic effects of MSCs (which mostly turned out false and stemmed from cross-contamination from adjacent tumor line cultures) led to a standardization of methods for evaluation of the MSC populations to be used in the abovementioned therapies. Purity of the MSC populations administered is crucial for the patient. Currently routine applications are based on a set of basic methods for MSC identification established in 2006 by the ISCT (Horwitz, E. et ah, “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement ”; Cytotherapy. 2006;8(4):315-7), based largely on the criterion of surface marker analysis, evaluation of MSC morphology, differentiation and ability to adhere to flat glass and plastic surfaces. However, these techniques have limited specificity and only guarantee a rough evaluation of a population.

Evaluation of MSCs in terms of their morphology and ability to adhere to plastic surfaces is an imprecise method, as dozens of different cell lines have been isolated that exhibit the same ability. On the other hand, the method of studying cell differentiation is time-consuming. The time required to properly evaluate multipotency according to procedures is at least three weeks just for the cultures. The proposed molecular method takes less than 5% of this time.

As the most accurate of the proposed methods, the evaluation of CD surface markers is a precise method for assaying MSCs, but in practice it is not without inherent flaws. Most importantly, it is based on reading surface markers. For this reason, the results of subsequent experiments may vary, depending on the culture duration, methodology and duration of cell detachment from the surface and the harshness of the reagents used for this purpose. This is due to the cells’ susceptibility to cell membrane damage and the resulting destruction of the conformation of the surface proteins being analyzed.

The nuclear and cellular gene expression assays in the proposed molecular considerations are largely free of this problem, as the detachment conditions do not affect gene expression. Furthermore, expression assays are characterized by a much higher accuracy of results (by several hundred times), owing to the selected genes identified due to their qualitative (negative or positive selection) or marked quantitative differences in expression, among other factors. Moreover, in molecular analyses there is no dependence on the fluorochromes conjugated to the same antibodies actually used, which would introduce additional disturbances and lower the cytometric assay sensitivity. Additionally, the differences stem from functional differences between the investigated genes; with surface markers, we only analyze the external layer, the approximate product of the mechanisms operating inside the cell. In molecular analyses, any gene can be assayed, often a transcription factor acting at the beginning of a signaling pathway; therefore, the assay is more sensitive even in terms of biology and allows a more rapid analysis of changes even before they materialize on the cell membrane (if they do at all).

Furthermore, cytometric methods and device settings across subsequent measurements are much more susceptible to qualitative discrepancies that affect the final results as compared to assays using RT-PCR; together with the effects of the culture and inter-individual variability, this supports the proposed molecular method.

To summarize, RT-PCR provides repeatable and stable data due to the use of references and results in the form of relative expression of the investigated genes.

Based on the findings documented by multiple research groups, the inventors also infer a strong similarity between MSC populations derived from different portions of the umbilical cord [Nagamura-Inoue, T., & He, H. (2014); Mennan et al. (2013)], as well as from other perinatal tissues (placenta, fetal membranes, chorionic villi) [Bieback, K., & Brinkmann, I. (2010); Kwon, A. et al. (2016)]. The identified similarities exist both at the morphological, phenotypic (including the presence of characteristic surface markers) [Wu, M., (2018); Schmelzer, E. et al. (2019)] and molecular levels. As a result, the inventors hypothesize that the developed panel series could be used successfully for the molecular analysis of MSCs derived from other perinatal tissues as well. The scientific information accrued so far on this issue suggests only inter-individual variability across the indicated populations and differences that do not affect their general characteristics. What is important, these are not applicable to such important parameters as the expression levels of genes responsible for pluripotency [Heo, J. (2016)] or the proposed MSC line markers.

Due to the lack of a molecular method to characterize MSC populations, there is a considerable market need for developing new diagnostic tools that would be more sensitive and less costly. This invention satisfies these expectations. Due to being based on strictly molecular and intracellular gene expression-based methods, the panel developed by the inventors to differentiate MSCs and perinatal tissue-derived cells from other lineages permits, under certain conditions, removing any reference to ISCT-approved marker panels routinely used for this purpose. The protocol developed by the inventors can be used successfully for the purpose of differentiating the expression profile of perinatal tissue-derived MSC lineage (via both positive and negative selection) from human pluripotent cells (hIPSCs) or select tumor lines originating from the 3 different germ layers at a molecular level.

The subject of the invention is a set of genes comprising at least one gene panel selected among: 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, IDOl, NANOG, POUF5F1, SOX2; for use in the determination of the pluripotent and teratogenic potentials of a mesenchymal cell (MSC) population or a perinatal cell population.

Preferably, the relative gene expression is as defined in Table 1 below.

Table 1

Preferably, mesenchymal cells are derived from the umbilical cord, bone marrow or adipose tissues; more preferably, mesenchymal cells are derived from Wharton’s jelly.

Preferably, the CDKN2A, CDH20, FLAND2, PDGFR-a genes are a subset of genes indicating a characteristic mesenchymal cell population expression profile.

Preferably, the ALOX15, CDH9, DRD4, ESM1, HEY1, NKX2-5 genes are a subset of genes indicating the lack of a teratogenic potential of an MSC population towards mesodermal lineage tumors.

Preferably, the FUT3, PROM1, COL2A1, FOXAl, MY03B genes are a subset of genes indicating the lack of a teratogenic potential of an MSC population for ectodermal lineage tumors. Preferably, the CLDN1, CPLX2, F0XA2, HNF1B, HNF4A, LEFTY1, POU4F1 genes are a subset of genes indicating the lack of a teratogenic potential of an MSC population for endodermal lineage tumors.

Preferably, the TDGF1, DNMT3B, IDOl, NANOG, POUF5F1, SOX2 genes are a subset of genes indicating the lack of a teratogenic potential of an MSC population for tumors of other origins.

The applicant is the first to address the need for a thorough analysis of MSCs and perinatal tissue-derived cells to differentiate them from lineages capable of pluripotency, carcinogenicity, as well as those originating from different germ layers.

Gene expression was calculated from the base-2 logarithm of the relative expression for mesenchymal cells versus the average expression for the control population of cells with the relevant characteristics (mesenchymal, mesodermal tumor, ectodermal tumor, endodermal tumor or pluripotent cells), which constitute a control population for a given panel, normalized for the reference genes: GAPDFl, F1PRT and ACTB, and is contained within the 95% confidence interval (Cl).

The preferable features of the invention are illustrated using the figures below, which supplement the information included in the embodiments.

Description of ffeures

Fig. 1 shows a comparison of gene expression in the expression panel characteristic for WJ- MSCs, selected for iPS cells and representative ectodermal (ZR-75-30, A-375), mesodermal (FIT-1080, MCF-7) and endodermal (NCI-H727, A-375) tumor lines. The positive selection of the mesenchymal cell investigated lines (MSC control) against the other investigated lines (tumor and human iPS) is shown.

Fig. 2 shows a comparison of the expression of genes characteristic for tumor lines of mesodermal origin selected against the other investigated populations. For clarity, the plot only includes the expression for the mesenchymal cell (MSC) line and the expression range for mesodermal tumors (FIT- 1080, MCF-7 lines).

Fig. 3 shows a comparison of the expression of genes characteristic for tumor lines of ectodermal origin selected against the other investigated populations.

For clarity, the plot only includes the expression for the mesenchymal cell (MSC) line and the expression range for ectodermal tumors (ZR-75-30, A-375 lines).

Fig. 4 shows a comparison of the expression of genes characteristic for tumor lines of endodermal origin selected against the other investigated populations. For clarity, the plot only includes the expression for the mesenchymal cell (MSC) line and the expression range for endodermal tumors (NCI-H727, A-549 lines).

Fig. 5 shows a comparison of the expression of genes characteristic for human pluripotent cell lines selected against the other investigated populations. For clarity, the plot only includes the expression for the mesenchymal cell (MSC) line and the expression range for IPS cells.

Examples

Materials and methods

Procedure 1 - Cell detachment and preparation for further procedures Equipment and materials

• Laminar flow cabinet

• Inverted microscope

• Incubator

• Centrifuge

• Culture dish with MSCs

• Medlab 3 mL Pasteur pipettes

• Corning sterile 15/50 mL Falcon tubes

• Ecolab Klercide 70/30

• Contec sterilesorb wipes

Reagents

• Biological Industries MSC NutriStem complete culture medium

• CSL Behring GmbH human albumin

• Life Technologies Tryple Select (IX)

• Fresenius Kabi sterile physiological saline

• Gibco Antibiotic-Antimycotic solution

Detailed procedure description:

1. At 4 days after the most recent passage (p2) of MSCs (if confluence was approximately 80%) in an incubator - 37°C, 5% CO2, in NutriStem™ complete culture medium, remove the supernatant from the culture dish.

2. Wash the culture dish with physiological saline.

3. Add 1 mL of Tryple (IX) enzyme solution / 75 cm² culture surface area into the culture dish.

4. Close the culture dish, stir gently to allow the liquid level in each layer to equalize and place in the incubator (37°C, 5% C02) for 5 minutes.

5. When this time has passed, check under the inverted microscope whether the cells have changed their shape to spherical and detached from the surface.

6. If the cells are not detached, return the culture dishes to the incubator and check after 5 more minutes.

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

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

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

10. Transfer the dish contents to sterile plastic Falcon tubes.

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

12. Transfer the culture dish contents to sterile Falcon tubes again.

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

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

15. Resuspend in an appropriate volume of culture medium for further procedures. Procedure 2 - Total RNA isolation from mesenchymal stem cells (MSCs); RNA purification, concentration measurement and quality control

Equipment and materials:

• Single channel pipettes

• Centrifuge

• Thermomixer C

• Vortex

• Sterile RNase-free tubes

• Sterile filter tips

• RNaseZap (Ambion, AM9780)

• Sterile RNase-free 20G (0.9 mm) 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)

• 2 M dithiothreitol (DTT)

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

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

Detailed procedure description:

1. Centrifuge the cells (no more than 5 x 10⁶) [5 minutes, RT, 1500 rpm] and remove the supernatant.

2. Add 350 pL of Buffer RTL with DTT to the cell pellet.

3. Mix thoroughly by vortexing or pipetting up and down to homogenize the mixture.

4. Subject the cell lysate to further homogenization by pipetting up and down or by using an RNase-free 20G needle and syringe (pipette the lysate up and down at least 5 times).

5. Vortex for 1 min.

6. Add 350 pL of 70% ethanol and mix thoroughly by pipetting up and down. Do not centrifuge.

7. Transfer 700 pL of the sample (including any precipitate that may have formed) to an RNeasy column (RNeasy spin column from the Qiagen RNeasy Plus Mini Kit) placed in a 2 mL collection tube.

8. Centrifuge for 30-60 seconds at > 8000 x^(> 10,000 rpm).

9. Remove the minicolumn, discard the flow-through and replace the minicolumn in the collection tube.

10. Add 350 pL of Buffer RW1 to the column. Close and centrifuge for 30 seconds at > 8000 x g (> 10,000 rpm). 11. Discard the flow-through as per step 10.

12. Add 80 pL of DNase I mixture directly onto the column bed. Incubate at room temperature for 15 min.

13. Add 350 pL of Buffer RW1 to the RNeasy spin column. Close and centrifuge for 30 seconds at > 8000 x g (³ 10,000 rpm).

14. Discard the flow-through as per step 10.

15. Add 500 pL of Buffer RPE to the RNeasy spin column. Close and centrifuge for 30 seconds at > 8000 xg(> 10,000 rpm).

16. Discard the flow-through as per step 10.

17. Add 500 pL of Buffer RPE. Close and centrifuge for 2 min at > 8000 xg- (> 10,000 rpm).

18. Transfer the RNeasy spin column to a new 2 mL collection tube. Gently close and centrifuge at maximum speed for 1 min.

19. Transfer the RNeasy spin column to a 1.5 mL RNase-free tube.

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

21. Centrifuge for 1 min at > 8000 g(³ 10,000 rpm).

22. Subject the eluate to further analyses or freeze at -80°C.

Procedure 3 - Total RNA concentration measurement and qualitative evaluation

Equipment and materials:

• 0.5-10 pL single channel pipette

• UV cabinet

• BioDrop KAP61 UV-VIS spectrophotometer

• Vortex

• Sterile RNase-free tubes (0.2, 0 5 and 1 5 mL)

• Sterile RNase-free filter tips, 10 pL

• RNaseZap (Ambion, AM9780)

Reagents:

• RNase-free water Detailed procedure description:

1. Thaw the RNA samples and keep them on ice. Transfer the tubes with the RNA to the BioDrop spectrophotometer.

2. Place the samples in the cabinet. Turn on the spectrophotometer.

3. Vortex the RNA samples before measuring concentration.

4. Clean the BioDrop measurement port before measurement. 5. Calibrate the BioDrop against a reference sample (RNase-free water).

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

• Pathlength: pLite 0.5mm

• Dilution Factor: 1.000

• Background: On

• Units: ng/pl

• Factor: 50.00

7. Perform measurements in duplicate for each sample.

8. RNA concentration is measured using a spectrophotometer at a wavelength of 260 nm. The A260/A280 ratio should be 1.9-2.1; A230/A260 should be above 1.8. These values indicate an appropriate RNA purity.

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

Equipment and materials:

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

• Refrigerated microcentrifuge with appropriate rotors and adapters

• Thermomixer C, block heater or dry bath

• Vortex

• Autoclave

• Microwave oven

• Sub-Cell (Mini or Wide Mini) agarose gel electrophoresis system (Bio-Rad)

• UV transilluminator or Chemidoc XRS+ system (Bio-Rad)

• Sterile RNase-free tubes, 0.2 mL

• Sterile RNase-free filter tips, 10 pL, 200 pL, 1000 pL

• RNaseZap (Ambion, AM9780)

Reagents:

• RNase-free water (extremely pure)

• DEPC (solution)

• DEPC-treated water

• Agarose powder (molecular grade)

• 50X (concentrated) TAE (Tris-acetate-EDTA) buffer

• Midori Green Advance (ABO)

• RiboRuler High Range RNA Ladder (#SM1821, Thermo Fisher Scientific)

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

Detailed procedure description: 1. Weigh the required amount of molecular-grade agar (depending on gel size and concentration). To obtain the recommended 1.5% gel, weigh 1.5 g agarose per 100 mL of IX TAE buffer.

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

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

4. Mix agar powder with the buffer manually.

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

6. Cool the solution to approximately 60°C.

7. Add Midori Green Advance to the dissolved agarose (5 pL/100 mL). Stir the solution gently and then pour the gel into the prepared casting tray with a comb.

8. Leave for 30 to 60 minutes at room temperature until solidified.

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

10. When the gel has solidified, transfer it to the Sub-Cell placed on ice.

11. Cover the gel with IX TAE buffer (up to 2-5 mm above the top surface 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 up and down or vortexing in order to ensure a homogeneous distribution of reagents inside the vials.

14. Prepare the ladder using the following ingredients: a. 2 pL of 2X loading dye, b. 2 pL of RiboRuler High Range RNA Ladder, c. 2 pL of RNase-free water.

15. Vortex and quickly centrifuge the sample.

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

17. Transfer to ice immediately and cool for 3 minutes. Then load the sample on the gel immediately.

RNA sample preparation and analysis:

1. Thaw the RNA samples on ice.

2. Mix the contents of the tubes by slowly pipetting up and down or vortexing in order to ensure a homogeneous distribution of reagents inside the vials.

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

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

5. Heat for 10 minutes at 70°C in a dry bath or block heater. 6. Transfer to ice immediately and cool for 3 minutes. Then load the sample on the gel immediately.

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

8. Visualize the samples using a UV transilluminator.

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

Equipment and materials:

• Single channel pipettes

• Cooling rack (4°C) for 0.2 mL tubes

• Refrigerated microcentrifuge with appropriate rotors and adapters

• Thermomixer C

• Vortex

• Autoclave

• Microwave oven

• Sub-Cell (Mini or Wide Mini) agarose gel electrophoresis system (Bio-Rad)

• UV transilluminator or Chemidoc XRS+ system (Bio-Rad)

• Sterile DNase-free tubes, 0.2 mL

• Sterile DNase-free filter tips, 10 pL, 200 pL, 1000 pL

• DNaseZap (Ambion, AM9780)

Reagents:

• Nuclease-free water (extremely pure)

• Autoclaved water

• Agar powder (molecular grade)

• 50X (concentrated) TAE buffer

• Midori Green Advance (ABO)

• GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific)

• GeneRuler 50 bp DNA Ladder (Thermo Fisher Scientific)

• 6X TriTrack 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 60 mM EDTA).

Detailed procedure description:

1. Perform steps 1-8 as per Procedure 1.

2. Place the Sub-Cell on ice. Level it. Fill up to the line with IX TAE buffer (in autoclaved water). 3. When the gel has solidified, transfer it to the Sub-Cell placed on ice.

4. Cover the gel with IX TAE buffer (up to 2-5 mm above the top surface of the gel).

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

6. Mix the contents of the tubes by slowly pipetting up and down or vortexing in order to ensure a homogeneous distribution of reagents inside the vials.

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

8. Vortex and quickly centrifuge the sample.

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

10. Transfer to ice immediately and cool for 3 minutes. Then load the sample on the gel immediately.

DNA sample preparation and analysis:

1. Thaw the DNA samples on ice.

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

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

5. Transfer to ice immediately and leave until the sample is loaded onto the gel.

6. Load the DNA samples and the appropriate GeneRuler Ladder prepared in the previous steps into the gel wells and run the electrophoresis for approximately 45 minutes, until the orange G in the loading dye (orange dye) has migrated approximately 2/3 of the gel. If the samples are not sufficiently separated, extend the electrophoresis by another 15 minutes.

7. Visualize the samples using a UV transilluminator.

Procedure 6 - Reverse transcription

The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) along with its reagents was used to perform the RT-PCR. The reaction was performed according to standard

Cl 000 thermal cycler protocols and in accordance with the RevertAid First Strand cDNA

Synthesis Kit manufacturer’s recommendations. The obtained cDNA samples were secured by placing in a -80°C freezer.

Procedure 7 - Real-time PCR amplification (RT-PCR)

Reagents

• Nuclease-free water

• 2x POWERUP SYBR® Universal Master Mix (Thermo Fisher Scientific) • Forward primer, 100 mM stock

• Reverse primer, 100 mM stock

• cDNA template

A Thermal Cycler Cl 000 with a CFX-96 module was used to perform the qPCR reactions. Primers were diluted 1:10 and 1 :25 in nuclease-free water. The reaction was performed in three technical replicates per sample, according to standard thermal cycler and the POWERUP SYBR® manufacturer protocols, 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 cycles of denaturation and elongation [95°C for 15 seconds and 60°C for 1 minute]. Additionally, a step to check the melting curve for the investigated genes was added for each investigated sample.

Procedure 8 - Primer design

All primers for PCR analyses were designed according to standard procedures known in the prior art using the following software: NCBI Primer BLAST, NetPrimer and NCBI Gene Base.

Embodiments

Example 1

Molecular characteristics of human Mesenchymal Stromal Cells (MSCs )

The cultured Wharton’s jelly-derived WJ-MSC cell lines (n=13) were harvested at early and late passages (p3 and plO, respectively), following the standards included in Procedure 1, and were subjected to RNA isolation according to Procedure 2.

The purity, quality and integrity of the obtained genetic material were measured in accordance with the recommendations provided in Procedures 3 and 4.

The isolated genetic material was used for the purposes of comparison with commercially available RNA from a pluripotent IPS line and six commercial (ATCC, Sigma Aldrich) tumor lines (ZR-75-30, A-375, HT-1080, A-549, MCF-7, NCI-H727) of different germ layer origins (the researchers selected two lines per each germ layer, having previously traced their origins based on detailed descriptions of the lines from their manufacturers).

After confirming the high quality of the obtained material, the RNA was subjected to reverse transcription according to Procedure 6. The single-stranded cDNA obtained by this method, completely free of genomic DNA (according to Procedure 5), was used for real-time PCR amplification (according to Procedure 7) using, among others, the primer sets designed based on the information included in Procedure 8 and calibrating these based on standard curves. A broad screening of literature in the fields of cytology and oncology allowed the identification and analysis of a number of single potentially specific markers for the particular populations to determine the nature of the cultured cells. Based on cumulative data, 8 genes were ultimately selected (PROM1, CDKN2A, FUT3, TDGF1, HER2, SOX9, B4GALNT1, TWIST1), which were used to differentiate between the populations, and a decision was made to extend the study to include a set of genes comprising the hPSC Scorecard™ (additional 89-gene profile).

Two reference genes were always used when developing the RT-PCR-based experiment (routinely GAPDH and HPRT, and GAPDH and ACTB for predefined plates). Reagent purity controls (- NTC) and RNA controls were loaded onto the plates; a reference MSC sample for interplate calibration was used as well. The collected data in the form of a multiplate experiment were analyzed using commercial Maestro 1.1 software. The experiment results, in the form of genes selected from the set, were divided into six sets of tumor panels (MSC, pluripotency, non-specific tumor panels and panels characteristic for all three germ layers: the endoderm, mesoderm and ectoderm) which differentiate the MSC populations from pluripotent lineages, using both the predefined plates mentioned above and independently designed primers

The obtained relative expression results for individual genes (calculated using the 2 ^DDa method approved by the scientific community and proposed in 2001 by Professor Livak’s research team in the article entitled: “Analysis of Relative Gene Expression Data Using RealTime Quantitative PCR and the 2 ^DDGI Method”) in the individual panels were normalized each time for the control sample (an MSC line from the PBKM cell bank and the commercial IPS, ZR- 75-30, A-375, HT-1080, MCF-7, A-549, NCI-H727 lines, respectively, depending on the panel), and a Maestro 1.1 software built-in algorithm was used for calibration and to eliminate the potential interplate differences in readouts.

The final set of genes within each of the panels proposed by the inventors are shown below:

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

• Mesodermal panel (ALOX15, CDH9, DRD4, ESM1, HEY1, NKX2-5)

• Ectodermal panel (FUT3, PROM1, COL2A1, FOXA1, MY03B)

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

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

The obtained results in the form of expression of the identified genes normalized for the control biological groups (averaged expression of these genes in the MSC line, IPS line, endodermal, mesodermal and ectodermal tumor lines) were exported to a separate spreadsheet and logarithmized (base-2 logarithm) each time. This operation was necessary in order to obtain symmetrical sample distributions. The deviations resulting from the analysis are shown in the plots as error bars, whose ends are located at the maximal and minimal expression values obtained for a particular gene in the biological group. 95% confidence intervals (Cl) were then calculated for each gene and control biological group in a given panel.

Due to the above, the “zero” value on the Y axis is a baseline for the evaluation of the multiple of difference in gene expression between the control biological group and the other investigated lines. The last operation was to calculate the confidence intervals for the investigated MSC lines, based on the obtained expression values for individual genes in the panels. Thus, the ranges (multiples of gene expression) characteristic for the investigated MSC lines were obtained.

The inventors also hypothesize that MSCs from all perinatal tissue populations will behave identically under the conditions described.

Example 2 - Panel characteristic for the MSC population

Using the panel developed by the inventors to differentiate MSCs from other lineages, including the pluripotent IPS line and 6 tumor lines (two lines characteristic for each germ layer), the average expression for the MSC biological group (n=13 and n=3) was compared with the other lines. For this purpose, the relative expression values calculated based on the approved “2- DDOT” method for individual genes versus the MSC line were analyzed. The inventors sought genes, the expression of which expression, calculated based on the MSC biological group with a 95% confidence interval, did not overlap with the expression in the other investigated lines, thus suggesting that they are potential characteristic markers of the investigated MSCs. Screening of over 97 genes demonstrated the existence of 4 such genes (CDKN2A, CDH20, HAND2, PDGFR-a). A clear graphic presentation of the results required logarithmization (base-2 logarithm) of the obtained relative expression values for the investigated samples (normalized for the reference genes: ACTB, GAPDH and HPRT); therefore, the “zero” value on the Y axis is a baseline for the evaluation of the multiple of difference in gene expression between the MSC biological group, which is the control population for this panel, and the other investigated lines.

Based on the obtained values, the inventors determined the gene expression ranges which indicate a high convergence with the MSC population (Table 2).

Table 2

The panel of genes characteristic for the MSC population developed by the inventors (Fig. 1) can be used successfully to differentiate the expression profile of the MSC line (via positive selection) from human pluripotent cells (hIPSCs) or tumor lines originating from the 3 different germ layers at a molecular level with a 95% confidence interval (Cl).

Example 3 - Panel characteristic for ectodermal tumor cell lines

The inventors then focused on selecting groups of genes that would allow unambiguous differentiation of MSC lines from the selected tumor lines at a molecular level. A list of genes which are regularly overexpressed in tumor cells was identified based on extensive literature screening. Aware that no characteristic tumor markers expressed in 100% of tumor cells have been discovered so far, the inventors decided to extend the selection panels to include genes characteristic for the individual germ layers (ectoderm, mesoderm and endoderm) from which the selected tumor lines originated. It is obvious that MSCs of mesodermal origin should not express any genes typical for lineages originating from the other layers, and a decision was made to prove this conclusively. Gene expression was again calculated from a base-2 logarithm of the relative expression for mesenchymal stromal cells versus the average expression for the control population of ectodermal tumor cells, which are the control population for this panel (Fig. 2), normalized for the reference genes: GAPDH, HPRT and ACTB. The plots shown in Fig. 2 are contained in the 95% confidence interval (Cl).

The tumor markers identified by the inventors (FUT3 and PROM1) differed to the highest extent from the MSC investigated samples in terms of expression, which is why they are included in this specific panel. However, every tumor line studied so far has had a significantly increased expression of these genes as compared to the MSC biological group. The other genes, such as COL2A1, FOXA1 and MY03B, are routinely expressed in cells originating from the ectodermal germ layer; therefore, their expression in the MSC samples is multiple times weaker as compared to the control tumor lines. Based on the obtained values, the inventors determined the gene expression range limits which indicate a high convergence with the MSC population (Table 3).

Table 3

The panel of genes characteristic for the ectodermal tumor lines developed by the inventors (Fig. 2) can be used successfully to differentiate the expression profile of the MSC line (via negative selection) from human tumor lines originating from the ectodermal germ layer at a molecular level with a 95% confidence interval (Cl).

Example 4 - Panel characteristic for mesodermal tumor cell lines

Another selection panel developed by the inventors enabled differentiation based on the expression of genes characteristic for the mesodermal-origin tumor lineage, selected against the MSC biological group. The markers identified by the inventors (ALOX15, CDH9, DRD4, ESM1, HEY1 and NKX2-5) differed to the highest extent from the MSC investigated samples in terms of expression, while remaining characteristic for the mesoderm (common lineage for both MSCs and the selected tumor lines originating from the mesodermal germ layer), which is why they are included in this specific panel. Despite the convergence resulting from a common origin, the MSC samples showed no expression of ALOX15, CDH9 and NKX2-5 genes, and for other genes the expression levels remained many times lower as compared to the averaged expression for the control tumor lines.

Based on the obtained values, the inventors determined the gene expression range limits which indicate a high convergence with the MSC population (Table 4). Table 4

The panel of genes characteristic for the mesodermal tumor lines developed by the inventors (Fig. 3) can be used successfully to differentiate the expression profile of the MSC line (via negative selection) from human tumor lines originating from the mesodermal germ layer at a molecular level with a 95% confidence interval (Cl).

Example 5 - Panel characteristic for endodermal tumor cell lines

The endodermal panel (Fig. 4), the last one from the group of selection panels related to genes characteristic for germ layers, enabled differentiating the populations based on the expression of genes characteristic for the endodermal-origin tumor lineage, selected against the MSC biological group. The markers identified by the inventors (CLDN1, CPLX2, EOMES, FOXA2, HF1B, HNF4A, LEFTY1 and POU4F1) differed to the highest extent from the MSC investigated samples in terms of expression, while remaining characteristic for the endoderm (characteristic for the selected tumor lines originating from the endodermal germ layer), which is why they are included in this specific panel.

Based on the obtained values, the inventors determined the gene expression range limits which indicate a high convergence with the MSC population (Table 5).

Table 5

The panel of genes characteristic for the endodermal tumor lines developed by the inventors (Fig. 4) can be used successfully to differentiate the expression profile of the MSC line (via negative selection) from human tumor lines originating from the endodermal germ layer at a molecular level with a 95% confidence interval (Cl).

Example 6 - Panel characteristic for the pluripotent cell population

The last panel proposed by the inventors (Fig. 5) can be used successfully as an index of the pluripotency of the investigated cells. Out of all the selected genes, six genes which are usually considered to be strongly associated with proliferation and maintenance of undifferentiated state were ultimately selected. These include: TDGF1, DMNT3B, IDOl, NANOG, POU5F1 and SOX2. However, the premise of this panel was to prove that the investigated MSC populations exhibit no pluripotent properties and can thus be safely used in therapies as a safe ATMP. The provided results clearly exclude the possibility of spontaneous, infinite replication in organisms, as well as pluripotency gene expression-mediated neoplastic transformation (Table 6).

Table 6

The developed pluripotency panel demonstrated that all the investigated MSC populations exhibit minimal expression (hundreds of times lower than in IPS cells) or no expression (as demonstrated for TDGF1 and SOX2 genes) of the selected pluripotency genes with a 95% confidence interval (Cl). Furthermore, the tumor lines were characterized by a markedly stronger expression of the DNMT3B, POU5F1 and SOX2 genes than the MSC lines which were of interest to the inventors, thus providing additional evidence supporting the lack of any teratogenic potential of mesenchymal cells.

Example 7 - Theoretical - Panels characteristic for MSC populations derived from different perinatal tissues

Lastly, the inventors propose an additional set of gene panels for MSC populations derived from other perinatal tissues, including: the placenta, chorionic villi, fetal membranes, amniotic fluid. The panel set is identical to the ones already presented and includes genes as per the panels below:

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

Mesodermal panel (ALOX15, CDH9, DRD4, ESM1, HEY1, NKX2-5) Ectodermal panel (FUT3, PROM1, COL2A1, FOXA1, MY03B) • Endodermal panel (CLDN1, CPLX2, EOMES, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1)

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

A comprehensive description of the procedure recommended by the inventors to perform the experiment, thus confirming the research hypothesis, is provided below.

Thus, the use of three (n=3) fresh MSC lines (three biological replicates) derived from the other perinatal tissues is recommended for comparative studies.

Harvest the MSCs at an early passage (p3), following the standards included in Procedure 1, and subject them to RNA isolation according to Procedure 2. Measure the purity, quality and integrity of the obtained genetic material in accordance with the recommendations provided in Procedures 3 and 4.

Use the isolated genetic material for the purposes of comparison with commercially available RNA from a pluripotent IPS line and six commercial (ATCC, Sigma Aldrich) tumor lines (ZR- 75-30, A-375, HT-1080, A-549, MCF-7, NCI-H727) of proven different germ layer origins. After confirming the high quality of the obtained material, subject the RNA to reverse transcription according to the procedure (according to Procedure 6). Use the single-stranded cDNA obtained this way, completely free of genomic DNA (according to Procedure 5), for real time PCR amplification (according to Procedure 7) using, among others, the primer sets designed based on the information included in Procedure 8 for the following genes: (PROM1, CDKN2A, FUT3, TDGF1, HER2, SOX9, B4GALNT1, TWIST1), as well as the sets of genes comprising the hPSC Scorecard™ (additional 89-gene profile). Based on the previous experiments, continue to use two reference genes (GAPDH and HPRT, or GAPDH and ACTB for predefined plates), as well as load reagent purity controls (- NTC), RNA controls and reference MSC samples (for GAPDH and HPRT genes) onto the plates for interplate calibration. Finally, collect all of the obtained data in the form of a multiplate experiment and analyze using the commercial Maestro 1.1 software.

Divide the experiment results, in the form of genes selected previously from the set, again into five sets of differentiating panels (MSC, pluripotency panels and panels characteristic for all three germ layers: the endoderm, mesoderm and ectoderm) which differentiate the MSC populations from pluripotent and teratogenic lineages, using both the predefined plates mentioned above and independently designed primers. Calculate the relative expression versus control biological groups (MSC lines from the PBKM cell bank and the commercial IPS, ZR- 75-30, A-375, HT-1080, MCF-7, A-549, NCI-H727 lines, respectively, depending on the panel), taking into account the expression values obtained from the MSC cells harvested from the other perinatal tissues and ultimately comparing these to the gene expression range limits determined in the previous embodiments which indicate a high convergence with the umbilical cord-derived MSC population.

This experiment may be applied with the same effect in the determination of the teratogenic potential and pluripotent properties of umbilical cord-derived mesenchymal cell populations, as well as cell populations derived alternatively from other perinatal tissues. Based on a broad review of scientific literature, the inventors are certain of the considerable similarity of cells originating from the mesenchymal lineage, especially in the context of their primordial nature, and the differences observed in the analyses will not cause different results on panels related to such fundamental issues as pluripotent capacity or germinal origin. The exemplary literature which confirms the similarity between umbilical cord-derived mesenchymal cell populations and cell populations derived from other perinatal tissues is as follows:

* Nagamura-Inoue, T., & He, H. (2014). Umbilical cord-derived mesenchymal stem cells: their advantages and potential clinical utility. World journal of stem cells , (5(2), 195.

* Mennan, C., Wright, K., Bhattacharjee, A., Balain, B., Richardson, J., & Roberts, S. (2013). Isolation and characterisation of mesenchymal stem cells from different regions of the human umbilical cord. BioMed research international , 2013.

* Bieback, K., & Brinkmann, I. (2010). Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy. World journal of stem cells, 2(4), 81.

* Kwon, A., Kim, Y., Kim, M., Kim, J., Choi, H., Jekarl, D. W., ... & Park, I. Y. (2016). Tissuespecific differentiation potency of mesenchymal stromal cells from perinatal tissues. Scientific reports, 6(1), 1-11.

* Wu, M., Zhang, R., Zou, Q., Chen, Y., Zhou, M., Li, X., ... & Chen, Q. (2018). Comparison of the biological characteristics of mesenchymal stem cells derived from the human placenta and umbilical cord. Scientific reports, 8(1), 1-9.

* Schmelzer, E., McKeel, D. T., & Gerlach, J. C. (2019). Characterization of human mesenchymal stem cells from different tissues and their membrane encasement for prospective transplantation therapies. BioMed research international, 2019.

* Heo, J. S., Choi, Y., Kim, H. S., & Kim, H. O. (2016). Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow, umbilical cord blood, placenta and adipose tissue. International journal of molecular medicine , 37(1), 115-125.

Claims

1. A set of genes comprising at least one gene panel selected among: CDKN2A, CDH20, HAND2, PDGFR-a; or ALOX15, CDH9, DRD4, ESM1, HEY1, KX2-5; or FUT3, PROM1, COL2A1, FOXA1, MY03B; or CLDN1, CPLX2, EOMES, FOXA2, HNF1B, HNF4A, LEFTY1, POU4F1; or TDGF1, DNMT3B, IDOl, NANOG, POUF5F1, SOX2; for use in the determination of the teratogenic potential of a mesenchymal cell (MSC) population or a perinatal cell population.

2. A set of genes for use according to claim 1, wherein the relative gene expression is as defined in Table 1 below.

Table 1

3. A set of genes for use according to claim 1 or 2, wherein mesenchymal cells are derived from the umbilical cord, bone marrow or adipose tissues.

4. A set of genes for use according to claim 3, wherein mesenchymal cells are derived from Wharton ’ s j elly .

5. A set of genes for use according to claim 1 or 2, wherein the CDKN2A, CDH20, HAND2, PDGFR-a genes are a subset of genes indicating a characteristic mesenchymal cell population expression profile.

6. A set of genes for use according to claim 1 or 2, wherein the ALOX15, CDH9, DRD4, ESM1, HEY1, KX2-5 genes are a subset of genes indicating the lack of a teratogenic potential of an MSC population for mesodermal lineage tumors.

7. A set of genes for use according to claim 1 or 2, wherein the FUT3, PROM1, COL2A1, FOXA1, MY03B genes are a subset of genes indicating the lack of a teratogenic potential of an MSC population for ectodermal lineage tumors.

8. A 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 a teratogenic potential of an MSC population for endodermal lineage tumors.

9. A set of genes for use according to claim 1 or 2, wherein the TDGF1, DNMT3B, IDO 1, POUF5F1, SOX2 genes are a subset of genes indicating the lack of a teratogenic potential of an MSC population for tumors of other origins.