阅读说明：本技术 从孵化的爬行动物卵分离用于生产生物人造皮肤和皮革的细胞 (Isolation of cells from incubated reptile eggs for production of bio-artificial skin and leather ) 是由 贝蒂娜·C·奥弗高 于 2018-04-05 设计创作，主要内容包括：本发明涉及一种通过在体外培养从孵化的爬行动物卵的绒毛尿囊膜分离出的细胞生产生物人造爬行动物皮革的方法。本发明允许在不存在传统爬行动物养殖和为获取其皮肤而捕杀爬行动物所涉及到的道德问题的情况下,生产爬行动物皮革。此外,本发明还允许生产那些不能大量作为皮肤产品的物种的皮革,例如,来自濒危物种的爬行动物皮革。(The invention relates to a method for producing biological artificial reptile leather by in vitro culturing cells isolated from the chorioallantoic membrane of hatched reptile eggs. The present invention allows the production of reptile leathers without the ethical problems involved in traditional reptile farming and the trapping of reptiles for the harvesting of their skin. Furthermore, the invention allows the production of leathers of species that cannot be used in large quantities as skin products, for example reptile leathers from endangered species.)

1. An in vitro method for obtaining cells for the production of reptile leather, characterized in that it comprises the following steps:

a. obtaining cells from the eggs of a hatched reptile, and

b. isolating and/or culturing keratin cells, fibroblasts, melanocytes, stem cells, or precursor cells.

2. The method according to any of the preceding claims, characterized in that the cells are isolated by using surface markers.

3. The method according to any of the preceding claims, characterized in that the cells are isolated by flow cytometry, for example by Fluorescence Activated Cell Sorting (FACS).

4. The method according to any of the preceding claims, characterized in that the cells are isolated by culturing a mixed culture of cells in a cell-specific medium to obtain a substantially pure culture of a specific cell type.

5. The method according to any of the preceding claims, characterized in that a substantially pure culture of one cell type is obtained by physically separating one type of cells from the other cells, e.g. by trypsinization.

6. A process according to any preceding claim, characterised in that the cells are isolated from the chorioallantoic membrane.

7. A method according to any preceding claim, wherein the cells are isolated from the allantois, the allantoic cavity, the amnion, the amniotic sac, the protein, the yolk or the yolk sac.

8. Method according to any one of the preceding claims, characterized in that the epidermal and dermal stem cells, mesenchymal stem cells, melanocytes and/or fibroblasts are isolated by culturing in a cell-specific medium.

9. A method according to any one of the preceding claims, characterized in that the keratin cells, the fibroblasts or the melanocytes are obtained by differentiation of stem cells or precursor cells.

10. The method of claim 5, wherein the fibroblasts are differentiated from mesenchymal stem cells.

11. The method according to any of the preceding claims, characterized in that the cells are cultured in the presence of at least one growth factor.

12. The method according to any of the preceding claims, characterized in that the cells are cultured in the presence of fibroblast growth factor, epidermal growth factor and/or connective tissue growth factor.

13. A method according to any preceding claim, wherein the cells are cultured on a feeder cell layer.

14. The method of any preceding claim, wherein the cell is not genetically modified.

15. A method according to any one of the preceding claims, characterised in that it comprises obtaining a substantially pure culture of keratin cells, fibroblasts and melanocytes.

16. The method according to any of the preceding claims, characterized in that a culture of one or more cell types is cryopreserved.

17. A method according to any one of the preceding claims, characterized in that the keratin cells, the fibroblasts and the melanocytes are obtained from one species.

18. Method according to any one of the preceding claims, characterized in that the keratin cells, the fibroblasts, the melanocytes are obtained from the same egg.

19. The method according to any of the preceding claims, characterized in that different cell types are isolated from different eggs of the same species.

20. The method according to any of the preceding claims, characterized in that different cell types are isolated from different eggs of different species.

21. The method according to any of the preceding claims, characterized in that the reptile is selected from the group consisting of: animals of the order alligator (alligator brachyodes and crocodile), the order testudinate (terrapin and turtle), lumbricus, lepidoptera (lizards and snakes), and the order rostral (lizards or coracoid).

22. The method according to any of the preceding claims, characterized in that the reptile is selected from the group consisting of: snake, alligator brachypodi, crocodile, lizard, turtle, terrapin, iguana, fly lizard, avocado, fossil dragon, champignon, lizard, gecko, king snake, water snake, python, dendroaspis, viper, rattlesnake, crocodile, alligator brachypodil, and rhesus alligator.

23. The method according to any of the preceding claims, characterized in that it further comprises growing a mixed culture of keratin cells, fibroblasts and melanocytes into a biological artificial reptile leather.

24. The method according to claim 23, characterized in that the cells are cultured on a carrier such as a fiber or mesh.

25. The method according to any of the preceding claims, further comprising transplanting the mixed culture of keratinocyte, fibroblast and melanocytes onto a reptile for skin repair.

26. The method according to any one of the preceding claims, further comprising culturing keratin cells, fibroblasts and/or melanocytes and obtaining reptile collagen from these cells.

27. The method according to claim 26, characterized in that it further comprises producing artificial reptile leather from the collagen obtained.

28. A bioartificial skin product obtained or obtainable by the method of any one of claims 1-23.

29. Use of a hatched reptile egg for isolating keratin cells, fibroblasts, melanocytes, stem cells or precursor cells.

30. An in vitro cell culture comprising keratin cells, fibroblasts, and melanocytes obtained by the method of any one of claims 1-23.

Technical Field

The present invention relates to a method for isolating cells for the production of bio-artificial skin and leather from hatched reptile eggs. The present invention allows the isolated cells to be used for the production of reptile skin and leather without the ethical problems involved in traditional reptile farming and the killing of wildlife for harvesting of their skin. The invention also allows the isolated cells to be used for the production of leather from species that cannot be used in large quantities as products, for example reptile leather from endangered species. In addition, the separated cells can be used for tissue regeneration to reduce leather waste caused by scar tissue formation of cultured animals, so that the animals used in the traditional culture can be reduced. The method of separating cells from the hatched eggs means that no negative effects will be exerted on the animal, unlike for example biopsies performed for the same purpose.

Background

The use of leather for making clothing and the like has been known for thousands of years. However, the use of leather obtained from traditional reptiles breeding or trapping and killing wildlife involves a number of moral problems. Traditional reptile breeding is common in countries without animal welfare legislation, and animal needs in cage environments, peer relationships, etc. are generally not considered. However, due to the wide demand for reptile leathers of various ages, it is not possible to ban the use of reptile leathers due to the wide consumer demand.

In view of this, it would be advantageous to be able to isolate cells for the production of reptile leather from hatched reptile eggs by an in vitro method, so that any ethical problems related to animal welfare and the killing of animals only for the purpose of obtaining the leather can be avoided. Furthermore, the number of traditionally farmed and slaughtered animals can be reduced by using the isolated cells for scarless tissue regeneration of animals wounded on farms.

The prior art does not show that cells for the production of reptile leather have been isolated from hatched reptile eggs.

Disclosure of Invention

The aim of the present invention is to isolate the cells from the eggs of the reptiles that have hatched, thus avoiding all the ethical problems involved in the traditional way of obtaining raw materials from biopsies or from animals slaughtered for the leather industry. The main aspect of the present invention relates to a method for isolating cells from the chorioallantoic membrane of hatched reptile eggs:

an in vitro method for obtaining cells for reptile leather production comprising the steps of:

-obtaining cells from eggs of a hatched reptile, and

-isolating and/or culturing keratin cells, fibroblasts, melanocytes, stem cells or precursor cells.

The cells are isolated from eggs of a hatched reptile which have revealed live, freshly hatched oviparous animals. One of the sources of cells in the eggs of hatched reptiles is the chorioallantoic membrane. Other possible sources of cells in the eggs of hatched reptiles include the allantois, the allantoic cavity, the amnion, the amniotic sac, proteins, the yolk and the yolk sac.

The stem cell may be a dermal stem cell or a mesenchymal stem cell.

The cells are isolated by culturing in a suitable cell culture medium in which the cells can be maintained in a stem or precursor cell state or differentiated into fibroblasts, keratinocyte and melanocytes or any other cell type required for the in vitro production of reptile leather, thereby isolating the cells of the invention. Alternatively, as described in the examples, fibroblasts and keratinocyte cells can be obtained directly from the chorioallantoic membrane and can be expanded into a substantially pure culture of these cells. It is contemplated that melanocytes may likewise be isolated from the chorioallantoic membrane or differentiated from precursor cells or stem cells.

Furthermore, to avoid the formation of scar tissue, cells isolated from hatched egg shells or chorioallantoic membranes of a particular reptile individual may be cryopreserved and later used for tissue regeneration of that particular individual in the event of skin damage in a breeding facility.

Another aspect of the present invention relates to a bio-artificial reptile leather obtained or obtainable by the method as described above.

Yet another aspect of the present invention relates to a garment or leather article comprising at least a portion of the bio-artificial reptile leather obtained or obtainable by the process described herein above.

Yet another aspect of the present invention relates to the use of isolated keratin cells, isolated dermal and mesenchymal stem cells, melanocytes or immortalized fibroblasts obtained from hatched reptile eggs for the in vitro production of a biological artificial reptile leather product.

In addition, isolated or differentiated fibroblasts, keratinocytes and melanocytes may be grown in vitro for the production of reptile collagen. According to the methods described in WO2013149083, WO 20142014201406 and WO2017003999, reptile collagen can be used for producing artificial reptile leather products.

Brief description of the drawings

FIG. 1 is a schematic representation of eggs from reptiles, source: (Biology and evolution of crocodilians, Gordon Grigg and David Kirshner, 2015).

Figure 2 shows keratinocyte cells isolated from the chorioallantoic membrane of reptile eggs.

FIG. 3 shows fibroblasts isolated from the chorioallantoic membrane of reptile eggs.

FIG. 4 shows real-time PCR, which shows high expression of the stem cell marker CD90 in cells isolated from the chorioallantoic membrane of reptile eggs.

FIG. 5 illustrates real-time PCR showing high expression of the stem cell marker CD166 in cells isolated from the chorioallantoic membrane of reptile eggs.

Detailed Description

Leather has been an important source of material for humans in various applications, such as clothing for bookbinding (e.g., shoes, jackets, hats, bags, belts) and furniture coverings, due to its durability and flexibility. Especially reptile leather is considered a luxury item. However, there is a need for ethical issues in killing wild animals and/or farm animals for the purpose of obtaining only animal skin.

The invention relates to the isolation of cells from the hatching eggs of reptiles for the production of artificial leather from cells isolated from the hatching eggs of reptiles entirely by an in vitro method.

Reptiles are herein meant to refer to the conventional understanding of the term "reptile". Reptiles, by this definition, are amnioces that are devoid of soft hair or feathers. This group includes today's (i.e., non-extinctive) turtles (turtles), alligators (crocodilians), snakes (snakes), vermilion (ampheibanians), lizards (lizards), and lizards (tuatara). Examples of representative reptiles include turtles (turtles), tortoises (tortoises), iguana (iguana), exendins (agamas), avocados (chameleons), kentrones (skinns), discoderms (animals), lizards (lizards), geckos (geckos), king snakes (boas), water snakes (anacondas), pythons (pythons), dendroaspis (mambas), vipers (vipers), vipers (adders), rattlesnakes (ratekes), crocodiles (crocodiles), alligators (alligators), and rhesus alligator (gavials).

The isolation of cells from incubated reptile eggs to produce a bioartificial reptile leather has the advantage over biopsy for isolation of desired cells that it does not have any negative impact on the animal. The isolated cells can then be used to produce a bio-artificial reptile leather solely by using in vitro methods, thereby avoiding any ethical issues relating to the killing of wildlife solely for the purpose of harvesting the skin of the animal and animal welfare issues relating to traditional wildlife breeding. Another advantage of isolating cells from hatched wild animal eggs is that cells can be isolated from endangered dead species without risk of injury to individual animals, thereby protecting the entire species. Yet another advantage is that cells isolated from the chorioallantoic membrane of a particular reptile individual can be cryopreserved. These cells can later be used for scarless tissue regeneration of the skin of animals suffering from skin wounds on farms. If scar tissue forms, a portion of the skin will have to be discarded and not used for luxury goods production. By scar-free tissue regeneration, less skin is discarded, and the number of animals used to produce a certain number of luxury goods can be reduced.

The cells isolated from the incubated reptile eggs according to the present invention may be used to produce artificial reptile leather that may be used in any manner that traditionally uses reptile leather, for example, for clothing, furniture, clothing or applications and accessories on furniture, and other reptile leather garments (including house wear).

The cells isolated from the hatching eggs of a reptile according to the present invention are preferably cells isolated from the vascularized chorioallantoic membrane obtained from the hatching eggs of a selected reptile. As described elsewhere herein, cells may also be obtained from other portions of the hatching reptile eggs. Preferably, the cells isolated are keratin cells (keratinocytes), dermal stem cells (dermal stem cells), mesenchymal stem cells (mesenchyme stem cells), melanocytes (melanocytes) or fibroblasts (fibroplast cells). Furthermore, the cells isolated according to the invention can subsequently be dedifferentiated and/or transdifferentiated from fibroblasts, keratinocytes, melanocytes, etc. into cells with stem cell characteristics, which can subsequently be (re) differentiated into cells suitable for use in vitro for wild animal leather production or tissue regeneration.

The dedifferentiation and/or transdifferentiation of the fibroblasts and/or fibroblast cell lines into cells with stem cell characteristics (e.g., progenitor cells) can be performed after isolation of the cells from the chorioallantoic membrane, and these progenitor cells can be further differentiated into cells suitable for the production of reptile leather in vitro. The present invention may also utilize dedifferentiation and/or transdifferentiation of keratin cells into their progenitor cells. The dedifferentiation and/or transdifferentiation of the present invention may be a revertin-mediated.

The cell isolation of the present invention may be based on chorioallantoic membranes obtained from any reptile from which leather may or may not be currently obtained, for example, the order alligator (alligator brachycarpus), turtles (terrapin and turtle), lepidoptera (lizard and snake), and coracoia (lizard or sphaedon puntatus).

Method of producing a composite material

The main aspect of the present invention relates to a method for isolating living cells from hatched reptile eggs:

-obtaining cells from the eggs of a hatched reptile, and

-isolating and/or culturing keratin cells, fibroblasts, melanocytes, stem cells or precursor cells.

Figure 1 shows the different components of an egg of a reptile, including the chorion, the allantoic cavity (allantoic sac), the amnion, the amniotic sac, the amniotic fluid, the umbilical cord, the yolk sac and the yolk.

The protein (3) is very visible at the two ends of the egg in the middle hatching period and is mainly positioned at the two ends of the egg, and the protein (3) provides a water source and gradually becomes less to form a rubber-like pad along with the development. The yolk contained in the yolk sac (6) and the embryo developing in its amniotic sac (2) are also very visible. Less obviously, the entire contents of the egg are surrounded by the membranous chorion (4). The embryo is fed by yolk through the yolk arteries and veins, which exit and enter the embryo through the umbilical cord. This approach reminds developing placental mammals of the manner in which they derive their nutrients from the maternal blood supply throughout the placenta. The allantois resembles a balloon that bulges out of the visceral layer to store waste. During early development, gaseous ammonia is excreted, but this is gradually replaced by urea, which is stored in the allantoic cavity (5). The allantois is initially small and gradually dilates until it envelops most of the egg contents. Combined with the chorion, pressing against the shell, forming the chorioallantoic membrane, which conducts O between the embryo and the cavity (cavity) atmosphere through the shell and the membrane₂And CO₂And (4) exchanging. As the embryo's oxygen demand increases, the chorioallantoic membrane gradually enlarges, forming and expanding an opaque band (1) around the equator of the egg. Gas is carried by the chorioallantoic arteries and veins that exit and enter the embryo through the umbilical cord. Egg laying, the embryo is on top of the yolk, only a few millimeters long, and the egg content is almost entirely yolk and protein. Yolk and proteins are gradually consumed as the embryo grows, occupying less and less space as the volume of the embryo and allantoic cavity increases (Biology and evolution of crocodilians, Gordon Grigg and David Kirshner, 2015).

In one embodiment, the cells of the invention are isolated from the chorioallantoic membrane of hatched reptile eggs. The cells may also be isolated from other parts of the hatching egg.

In another embodiment, the reptiles referred to herein are endangered species and/or are selected from the group consisting of reptiles: animals of the order alligator (alligator brachyodes and crocodile), the order Testudinate (terrapin and turtle), the order Lepidoptera (lizard and snake), and the order Cereus (lizard or Cereus).

According to the method of the present invention, cells are isolated from different individuals of a reptile; alternatively, according to preferred embodiments, the cells are isolated from one and the same individual of a reptile.

In a preferred embodiment, the cell is not genetically modified.

In a preferred embodiment, cells from the chorioallantoic membrane may be propagated and stored for later use, thereby eliminating the need to collect hatching eggs each time the bioartificial reptile is produced. In addition, cells obtained from hatching the eggs of a reptile may be stored as a source of donor cells for the individual, e.g., for repairing damage to the reptile skin of the animal.

Method for manufacturing reptile leather

In one embodiment, reptile leather can be produced by culturing fibroblasts, keratinocyte and melanocytes and growing these cells into skin. To increase the strength of the skin or leather, these cells may be grown on a fibrous or mesh support. In addition, colorants may be added to make a reptile image on the skin. WO2016073453 describes a method of enhancing leather by growing cells into fibers or networks.

In another embodiment, the isolated reptile fibroblasts, keratinocyte cells, and melanocytes are grown in vitro to produce collagen. The collagen may be harvested, for example, by the methods described in WO2013149083, WO 20142014201406 and WO2017003999, and processed into artificial reptile leather.

Keratinocyte, dermal stem cell, mesenchymal stem cell, melanocyte and/or immortalized fibroblast

Stem cells (e.g., dermal and mesenchymal stem cells), keratinocytes, melanocytes, and/or fibroblasts, as described herein, can be isolated by culturing in a cell specific medium.

In one embodiment, the epidermal stem cells and dermal stem cells are isolated by culture.

Keratinocyte cells were isolated by culturing in conditioned CnT-prime epithelial cell culture medium (CellnTec) with or without a feeder layer of TC-treated cell culture plates, to which 10% chelated FBS was added.

Fibroblasts can be isolated by culturing in CnT-prime fibroblast culture medium (CellnTec) in TC-treated cell culture plates.

Mesenchymal stem cells can be isolated by culturing in DMEM/F12(1:1), 20ng/mLEGF, 40ng/mL bFGF, 2% B27 in cell culture plates that are not TC-treated, wherein the cells form free-floating spheres in the cell culture plates. The culture may also be a culture on TC treated plates, where the cells attach to the culture plate, then form spheres, and are released by adherent cells.

Mesenchymal stem cells can be differentiated into fibroblasts and keratinocytes, as well as other cell types, according to methods known in the art. Differentiation of mesenchymal stem cells into fibroblasts requires addition of connective tissue growth factor in growth medium (e.g., DMEM/F12, 2% B27, 20 μ g/mL EGF, 40 μ g/mL FGF).

By physically separating cells during culture, for example, by taking advantage of the varying degrees of adhesion between cells and their growth surfaces, certain cells can be grown into substantially pure cultured cells.

Culture medium

The following table gives a non-limiting list of media names and components that can be used for the isolation and cultivation according to the invention.

Examples of the invention

Preparation of Mixed cell populations from chorioallantoic Membrane

Chorioallantoic membranes were obtained from freshly hatched eggs of the Alligator brevis (Alligator mississippiensis) according to the method previously described by Kjelland kramer, Avian Biology Research 5(3), 2012.

Once exposed, the reptiles sample the hatched eggs by using sterile forceps to collect vascularized chorioallantoic membranes from the interior of the eggs. To minimize bacterial contamination, the chorioallantoic membranes were washed by transferring to tubes containing 25mL of DMMEM/F121: 1, 10% FBS, 50. mu.g/mL gentamicin, 1% PSA (penicillin streptomycin amphotericin B), 0.25. mu.g/mL amphotericin B and vortexing. The washing process was repeated 3 times in total by transferring the chorioallantoic membrane to a tube with fresh wash medium as described above.

Then, the chorioallantoic membrane was digested in 25 mM Accotase at 31 ℃ for 30-60 minutes, and the tube was gently shaken every 5 minutes. Accutase was aspirated and transferred to tubes containing CnT Prime epithelial cell medium (Celln Tech) containing 10% chelated FBS, 1% penicillin/streptomycin/amphotericin B. The cell solution was filtered sequentially through 100. mu.M, 70. mu.M and 40. mu.M cell filters. Cells were centrifuged at 300Xg for 10 min and the supernatant removed. The pelleted cell pellet was resuspended in conditioned Cnt Prime epithelial cell culture medium (Celln Tech) containing 10% chelated FBS, 1% penicillin/streptomycin/amphotericin B. To avoid cell culture infections, other antibiotics and antifungal agents may also be used, for example gentamicin, nystatin.

At this time, a portion of the cells were cryopreserved with 10% DMSO/20% FCS/70% CnT-prime, 10% FBS.

To isolate the keratinocyte cells, the cell solution was seeded on TC treated cell culture plates without feeder layer with the condition CnT Prime epithelial cell medium (CellnTech) containing 10% chelating FBS, 1% penicillin/streptomycin/amphotericin b (psa). Cell cultures were incubated at 31 ℃ with 5% CO₂Is carried out byAnd (5) culturing.

After 7-10 days, cells appeared, see FIG. 2 (species: Alligator brachycarpus).

The chorioallantoic membrane treated with Accutase was transferred to tubes containing 25mL of DMEM containing 0.2-1% collagenase I. The tubes were incubated overnight at 31 ℃ for digestion. The next day, tubes were retrieved and 25mL of DMEM/F12(1:1), 10% FBS, 1% PSA was added to the cell solution containing the digested chorioallantoic membrane. The cell solution was filtered sequentially through 100. mu.M, 70. mu.M and 40. mu.M cell filters. The cells were centrifuged at 300Xg for 10 minutes and the supernatant removed.

At this time, a portion of the cells were cryopreserved without isolating the specific cell type (using 10% DMSO/20% FCS/70% cell-specific medium), and the remaining cells were used for culture.

To isolate fibroblasts, the pellet was resuspended in CnT-prime fibroblast medium, 10% FBS, 1% PSA and seeded onto TC-treated cell culture dishes. At 31 deg.C, 5% CO₂Next, the cells were cultured. Fig. 3 shows fibroblasts from alligator americana.

During subsequent subcultures, cells can be detached using trypsin or dispase (dispase), leaving other more adherent cell types still attached to the dish, while fibroblasts will detach more rapidly, which will allow for near 100% subculture of fibroblasts.

To isolate mesenchymal stem cells, the cell pellet was resuspended in DMEM/F12(1:1), 20ng/mL EGF, 40ng/mL bFGF, 2% B27, 1% PSA and plated onto cell culture dishes that were not treated with TC. At 31 deg.C, 5% CO₂Next, the cells were cultured. Mesenchymal stem cells will form spheres and proliferate in the form of spheres, which can be isolated by removing the medium with the spheres and subsequent centrifugation. The removal of certain other cell types will be due to their attachment to the culture dish, which is discarded after the removal of the medium using the spheres.

Detection of the Presence of mesenchymal Stem cell markers by real-time PCR

Cell resuscitation

Frozen vials containing cell suspensions from digested allantoic chorionic membrane obtained from alligator americana (genus alligator) as described above were thawed at Room Temperature (RT). Once thawed, 1mL of complete medium (Dulbeccos Modified Eagle Medium (DMEM; Euroclone, Italy)) + 10% heat-inactivated fetal bovine serum (FBS, Euroclone) and 1% penicillin/streptomycin (P/S; Euroclone) were slowly added to the flask from the 31 ℃ environment. The cells were transferred to a tube containing 5mL of complete medium.

Centrifuge at 1200rpm for 4 minutes at room temperature. The supernatant was removed, 1mL of phosphate buffer (PBS; EuroClone) was added, and the cell pellet was washed.

A portion of the cell suspension was cell counted using a cell counter (Countess II, Thermo Fisher Scientific, Waltham, MA, USA) resulting in 5 cells/mL at 3.05X10 e. The remaining cells were centrifuged at 1200rpm for 4 minutes at room temperature.

As a control, vials containing fibroblasts obtained from reptiles belonging to the alligator family were thawed using the same method as described above. The fibroblast control sample was cell counted and found to be 6.4x10e5 cells/mL.

Total RNA isolation

Total RNA was isolated from control and assay samples using a total RNA purification kit (Norgen Biotek, Thorold, Canada) according to the manufacturer's protocol. mu.L of Buffer RL was added to the cell pellet and then transferred to a gDNA removal column assembled onto a collection tube.

The lysate was centrifuged at 140000rpm for 1 minute at room temperature. The effluent was collected and added to 210. mu.L of absolute ethanol, and then loaded onto an RNA purification column assembled into a new collection tube. The solution was centrifuged at 6000rpm for 1 minute at room temperature.

The column was washed by adding 400. mu.L of washing Solution A (Wash Solution A) to the column. The column was centrifuged at 14000rpm for 1 minute at room temperature. The washing step was repeated 3 times.

The total RNA collected was diluted by adding 20. mu.L of eluent A (elution Solution A) to each column and centrifuged at 2000rpm for 2 minutes, followed by 14000rpm for 1 minute at room temperature.

Using NanoDrop^TMND-1000(Thermo Fisher Scientific) measures RNA quality and concentration of samples and controls. The total RNA of the sample was found to be 187 ng/. mu.l (260/280 ═ 1.95; 260/230 ═ 1.81). Control total RNA was found to be 258.6 ng/. mu.l (260/280 ═ 2.06; 260/230 ═ 1.91).

First Strand cDNA Synthesis

According to SensiFAST^TMcomplementary DNA (cDNA) was synthesized by cDNA synthesis kit (Bioline, Singapore). For samples and controls, 500ng of total RNA was reverse transcribed in a reaction volume of 20. mu.L. Each reaction contained 4. mu.L of 5XTransAmp buffer, 1. mu.L of reverse transcriptase and 15. mu.L of RNA (500 ng each for samples and controls). The reaction was carried out in a LifePro ThermalCycler (bio Technology), bosch technologies, china, using the following procedure: primer annealing at 25 ℃ for 10 min, reverse transcription at 42 ℃ for 15 min, and enzyme inactivation at 85 ℃ for 5 min. Their cDNAs thus generated were stored at-20 ℃ until real-time PCR was performed.

Real-time PCR

Real-time PCR was performed on Rotor-Gene 3000(Corbett Research, Sydney, Australia). A total of 1.5. mu.L of cDNA was added to a final reaction volume of 15. mu.L. The reaction mixture was composed of 7.5. mu.L of 2 XSensiFAST^TM

No-ROX mixture (Bioline), 1.2. mu.L each of 10. mu.M forward and reverse primers, and 3.6. mu.L of water. The following PCR procedure was used: 95 ℃ for 2 minutes, then 45 cycles: of these, 95 ℃ for 5 seconds, 60 ℃ for 10 seconds, and 72 ℃ for 20 seconds. Samples and controls were tested for CD90 and CD166, which are distinctive signatures of mesenchymal-derived stem cells. Beta-actin was used as an internal reference to ensure the same starting conditions and to normalize the relative expression data.

The primer sequences used were: CD90 forward primer: AGCAAGGACGAGGGCACCTACA, reverse primer: TGGGAGGAGATGGGTGGGGAAT, respectively; CD166 forward primer: TCAAGGTGTTCAAGCAACCA, reverse primer: CTGAAATGCAGTCACCCAAC, respectively; β -actin forward primer: TGGTGGGCATGGGTCAGAAGGA, reverse primer: ATGCCGTGCTCGATGGGGTACT are provided.

To quantify mRNA transcription, the relative 2 Δ Δ Ct method [ Pfaffl ] was used. Relative quantitation is commonly used to compare the expression levels of genes in different samples. In studying gene expression, the number of gene transcripts of interest needs to be normalized to the variation in sample quality and number between different samples. To ensure the same starting conditions, relative expression data were normalized using β -Actin (ACTB) as an internal reference.

The results show that relative expression of CD90 (also referred to as YHY1) was about 4300-fold higher compared to control (fibroblasts) and housekeeping gene β -actin (fig. 4); relative expression of CD166 (also known as ALCAM) was about 580-fold higher compared to control (fibroblasts) and housekeeping gene β -actin (fig. 5); this demonstrates the presence of mesenchymal-derived stem cells.

Reference to the literature

Kjelland et Kraemer,Avian Biologiy Research 5(3),2012

Chang et al,Int J Dev Biol.2009；53(5-6):813–826

Pfaffl,MW.A new mathematical model for relative quantification inreal-time RT-PCR.NucleicAcids Res.2001；29(9):e45.