Reprogramming medium and method for reprogramming chinchilla somatic cells into pluripotent stem cells

Abstract

Embodiments of the present invention relate to the technical field of cell reprogramming, and disclose a reprogramming culture medium and a method for reprogramming chinchilla somatic cells into pluripotent stem cells. Among them, the culture medium includes culture medium I and culture medium II; culture medium I is a DMEM culture medium containing ≥5 v / v% serum substitute, ≥0.2 v / v% Nucleosides, ≥0.2 v / v% GlutaMax, 0.1 mM - 10.0 mM NEAA, 0.02 mM - 1 mM 2-mercaptoethanol, 1000 - 3000 U / mL LIF, 0.3 μM - 10 μM PD0325901, and 1 μM - 10 μM CHIR-99021; culture medium II is a complete culture medium for C57BL / 6 mouse embryonic stem cells. The method includes: reprogramming chinchilla somatic cells by transfecting transcription factors, and culturing the reprogrammed chinchilla somatic cells with culture medium I and culture medium II in sequence after transfection. By applying the technical solution of the present invention, chinchilla IPSCs can be successfully obtained.

Description

Technical Field

[0001] The embodiments of the present application relate to the technical field of cell reprogramming, and in particular to a reprogramming medium and a method for reprogramming chinchilla somatic cells into pluripotent stem cells. Background Art

[0002] Pluripotent stem cells are a type of stem cells with the potential to differentiate into multiple cell types. Due to their self-renewal and differentiation potential, they have broad application prospects in the fields of regenerative medicine, disease research, and drug screening. Among them, induced pluripotent stem cells (iPSCs) are pluripotent stem cells generated by reprogramming somatic cells. During the cell reprogramming process, differentiated somatic cells are reprogrammed under specific conditions and restored to a totipotent state or form an embryonic stem cell line. When reprogramming cells, iPSCs usually need to be cultured and induced with a reprogramming medium. Currently, conventional reprogramming media mainly include DMEM basal medium and nutrients, etc.

[0003] At present, there are already some literatures and reports on the reprogramming cases of animal cells such as humans, mice, pigs, cows, and sheep. In the reprogramming of animal cells, iPSCs can be successfully induced and cultured with a conventional reprogramming medium. Based on the reported reprogramming cases of animal cells, researchers expect to obtain chinchilla iPSCs by reprogramming chinchilla somatic cells. Chinchillas are small and obese, with long and fluffy hair at the end of their tails. They are chipmunk-like animals and are named after their soft and dense body hair. Chinchilla somatic cells are reprogrammed to obtain chinchilla iPSCs, and then the chinchilla iPSCs are directionally differentiated in vitro. Finally, a chinchilla sac structure is obtained in vitro. The chinchilla hair grown from the chinchilla sac structure can be used as green fur. The method of obtaining chinchilla hair by directionally differentiating chinchilla somatic cells through chinchilla iPSCs has important scientific research potential.

[0004] However, the animals for which reprogrammed cells have been achieved have a large species difference from chinchillas, and the species closest to chinchillas only belong to the same order. It is difficult to obtain chinchilla iPSCs from chinchilla somatic cells through the existing technology. Summary of the Invention

[0005] To solve the above problems, the invention object of the first aspect of the present application is to provide a reprogramming medium. Through the induction culture of Medium I and Medium II, chinchilla iPSCs can be successfully obtained, and it is beneficial to improve the cell reprogramming efficiency.

[0006] The invention object of the second aspect of the present application is to provide a method for reprogramming chinchilla somatic cells into pluripotent stem cells. The steps are simple. Medium I and Medium II are used to culture and induce cells in sequence, which is beneficial to obtain high-quality chinchilla iPSCs while improving the cell reprogramming efficiency.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the reprogramming medium of the present invention comprises Medium I and Medium II;

[0009] The Medium I is a DMEM medium containing ≥5 v / v% serum substitute, ≥0.2 v / v% Nucleosides, ≥0.2 v / v% GlutaMax, 0.1 mM - 10 mM NEAA (Nonessential Amino Acids), 0.02 mM - 1 mM 2-mercaptoethanol, 1000 - 3000 U / mL LIF (leukemia inhibitory factor), 0.3 μM - 10 μM PD0325901, and 0.1 μM - 10 μM CHIR-99021; the Medium II is a complete medium for C57BL / 6 mouse embryonic stem cells.

[0010] In some implementation modes, the complete medium for C57BL / 6 mouse embryonic stem cells contains 5 v / v% - 30 v / v% FBS, 0.2 v / v% - 3 v / v% Glutamine, 0.2 v / v% - 3 v / v% NEAA, 0.01 - 0.1 v / v% LIF, and 0.05 v / v% - 0.3 v / v% 2-mercaptoethanol.

[0011] In some implementation modes, both Medium I and Medium II comprise 0.1 v / v% - 3 v / v% penicillin / streptomycin double antibody (penicillin / streptomycin double antibody).

[0012] In some implementation modes, the serum substitute in Medium I is 5 v / v% - 20 v / v%, the Nucleosides is 0.2 v / v% - 3 v / v%, and the GlutaMax is 0.2 v / v% - 3 v / v%.

[0013] In a second aspect, a method for reprogramming chinchilla somatic cells into pluripotent stem cells, using the above-mentioned reprogramming medium, comprises the following steps:

[0014] Chinchilla somatic cells are transfected with transcription factors by reprogramming, and after the reprogramming transfection of chinchilla somatic cells, they are cultured successively with Medium I and Medium II.

[0015] In some implementation modes, the transcription factors are Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1, and L-Myc.

[0016] In some implementations, it is cultured in Medium I for 2 to 5 days.

[0017] In some implementations, it is cultured in Medium II for ≥5 days.

[0018] In some implementations, when culturing in Medium II, the cells need to be cultured on a feeder layer.

[0019] In some implementations, after the chinchilla somatic cells are cultured in the dermal cell medium, they are then reprogrammed by transfecting transcription factors. Among them, the dermal cell medium contains 15 v / v% - 25 v / v% FBS, 0.4 v / v% - 1 v / v% penicillin / streptomycin double antibody, and the balance is DMEM high-glucose medium.

[0020] Based on the above technical solutions, the present invention has the following technical effects:

[0021] 1. The reprogramming medium provided by the present invention can reprogram and differentiate chinchilla somatic cells (dermal cells) into induced pluripotent stem cells. It requires fewer reagents and has a low cost.

[0022] 2. The method for reprogramming chinchilla somatic cells into induced pluripotent stem cells provided by the present invention realizes the reprogramming of chinchilla somatic cells for the first time. First, the dermal cells are reprogrammed into pluripotent stem cells by co-starting the transfection of reprogramming factors with reagents such as LIF, PD0325901, and CHIR-99021 in Medium I, and then changing to Medium II can effectively promote the reprogramming of chinchilla somatic cells (dermal cells). When used in combination with Medium I and Medium II, the differentiation efficiency is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a microscope image of chinchilla IPS in Example 1 of the present invention, with a scale bar of 500 μm in the figure.

[0024] Figure 2 It is a microscope image of chinchilla IPS in Example 2 of the present invention, with a scale bar of 500 μm in the figure.

[0025] Figure 3 It is a microscope image of chinchilla IPS in Comparative Example 1 of the present invention, with a scale bar of 500 μm in the figure.

[0026] Figure 4 It is a microscope image of chinchilla IPS in Comparative Example 2 of the present invention, with a scale bar of 500 μm in the figure.

[0027] Figure 5 It is a microscope image of chinchilla IPS in Comparative Example 3 of the present invention, with a scale bar of 500 μm in the figure.

[0028] Figure 6 Microscopic image of the chinchilla IPS of Comparative Example 4 of the present invention. The scale bar in the figure is 500 μm.

[0029] Figure 7 Gene test result graph of Oct3 / 4 of the chinchilla IPS of Example 2 of the present invention.

[0030] Figure 8 Gene test result graph of Sox2 of the chinchilla IPS of Example 2 of the present invention.

[0031] Figure 9 Gene test result graph of KIf4 of the chinchilla IPS of Example 2 of the present invention.

[0032] Figure 10 Gene test result graph of C-myc of the chinchilla IPS of Example 2 of the present invention.

[0033] Figure 11 Gene test result graph of Nanog of the chinchilla IPS of Example 2 of the present invention.

[0034] Figure 12 Immunofluorescence staining result graph of the chinchilla IPS of Example 2 of the present invention. Detailed implementation mode

[0035] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below in conjunction with specific embodiments. The present invention provides preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0036] Before further describing various embodiments of the compounds / compositions and methods of the present disclosure in more detail through exemplary descriptions, examples, and results, it should be understood that the embodiments of the present disclosure are not limited to the details of the methods and compositions described in the following description in their applications. The descriptions provided herein are for illustrative purposes only and are not to be construed in a limiting sense. The inventive concept of the present disclosure can have other embodiments or can be practiced or implemented in various ways. Therefore, the language used herein is intended to give the broadest scope and meaning; and the embodiments are intended to be exemplary and not exhaustive, and are not intended to limit the present disclosure to these specific embodiments. Moreover, it should be understood that the wording and terms used herein are for descriptive purposes and should not be considered restrictive unless otherwise stated. In addition, in the following detailed description, many specific details are listed to provide a more thorough understanding of the present disclosure.

[0037] However, it will be apparent to those of ordinary skill in the art that embodiments of the present disclosure can be practiced without these specific details. In other instances, features known to those of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. It is intended that all alternatives, substitutions, modifications, and equivalents that are apparent to those of ordinary skill in the art be included within the scope of the present disclosure. All compounds / compositions and their preparation methods, applications, and uses disclosed herein can be prepared and implemented without undue experimentation in accordance with the present disclosure.

[0038] Accordingly, although the compounds / compositions and methods of the present disclosure have been described in terms of specific embodiments, it will be apparent to those skilled in the art that changes can be made to the formulations, compounds or compositions, and / or methods and to the steps or the order of steps of the methods described herein without departing from the spirit and scope of the inventive concept of the present disclosure.

[0039] As used herein, any reference to "an embodiment" or "embodiments" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The phrase "in one embodiment" appearing multiple times in the specification is not necessarily all referring to the same embodiment.

[0040] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular.

[0041] The following further details the present application.

[0042] The reprogramming medium of the present invention comprises Medium I and Medium II;

[0043] Medium I contains ≥5 v / v% serum substitute, ≥0.2 v / v% Nucleosides, ≥0.2 v / v% GlutaMax, 0.1 mM - 10.0 mM NEAA, 0.02 mM - 1 mM 2-mercaptoethanol, 1000 - 3000 U / mL LIF, 0.3 μM - 10 μM PD0325901, and 0.1 μM - 10 μM CHIR-99021, with the balance being DMEM medium.

[0044] In some specific embodiments shown, the addition amount of the serum substitute is preferably 5 v / v% - 20 v / v%, the addition amount of Nucleosides is preferably 0.2 v / v% - 3 v / v%, and the addition amount of GlutaMax is preferably 0.2 v / v% - 3 v / v%.

[0045] Specifically, the serum substitute provides necessary nutrients for the induced culture of chinchilla somatic cells and can support the growth and directed differentiation of chinchilla somatic cells. NEAA provides non-essential amino acids required for the growth of chinchilla somatic cells and can also balance the pH value of the growth environment of chinchilla dermal cells to ensure that reagents such as PD0325901 and CHIR-99021 synergistically play a role in promoting the initiation of cell reprogramming.

[0046] Medium II is the purchased HyCyte TM Complete medium for C57BL / 6 mouse embryonic stem cells, and the concentration of the additives it provides can be adjusted as needed.

[0047] In a preferred embodiment, the complete medium for C57BL / 6 mouse embryonic stem cells contains 5 v / v% - 30 v / v% FBS, 0.5 v / v% - 3 v / v% Glutamine, 0.5 v / v% - 3 v / v% NEAA, 0.01 - 0.1 v / v% LIF, and 0.05 v / v% - 0.3 v / v% 2-mercaptoethanol.

[0048] Specifically, Medium II can promote the reprogramming efficiency of chinchillas, enabling more than 80% of chinchilla somatic cells (dermal cells) to be reprogrammed into induced pluripotent stem cells within a cycle of 18 - 20 days.

[0049] In a preferred embodiment, both Medium I and Medium II contain 0.1 v / v% - 3 v / v% penicillin / streptomycin double antibody, effectively protecting chinchilla somatic cells from microbial invasion.

[0050] The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to the present invention uses the above-mentioned reprogramming medium and includes the following steps:

[0051] Chinchilla somatic cells are transfected with transcription factors during reprogramming. Specifically, chinchilla dermal cells can be preferably used as chinchilla somatic cells. Before reprogramming transfection, chinchilla dermal cells are inoculated into six-well plate A and labeled as well A. On the day after the chinchilla dermal cells are inoculated into well A, the dermal cell medium is replaced. The dermal cell medium contains 15 v / v% - 25 v / v% FBS, 0.4 v / v% - 1 v / v% penicillin / streptomycin double antibody, and the balance is DMEM high-glucose medium.

[0052] Reprogramming transfection of transcription factors is performed on dermal cells in pore A. In some specific embodiments, the transcription factors include Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1, and L-Myc. For example, for transcription factor transfection, the Epi5™ Episomal iPSC Reprogramming Kit can be used. Add 1 μL of each of the two tubes of plasmids, namely Epi5™ Reprogramming Vectors (Tube A) and Epi5™ p53&EBNA Vectors (Tube B), into pore A. Then, the dermal cells in pore A after transcription factor transfection are cultured. In a preferred embodiment, the dermal cells in pore A can be cultured in an incubator for 18 h to 24 h, specifically, such as 18 h, 20 h, or 24 h, and the culture temperature is 37°C.

[0053] The dermal cells in pore A are cultured by replacing the medium with Medium I for 2 to 5 days, with the medium changed daily, until the cell confluence in culture pore A reaches 100%. Medium I promotes the rapid programming of dermal cells in pore A and reduces the apoptosis of dermal cells in pore A while the cells are being programmed.

[0054] The dermal cells in pore A are passaged and transferred to six-well plate B. Specifically, when the cells in pore A are confluent, the dermal cells in pore A are passaged at a ratio of 1:3 into six-well plate B, and feeder layer cells have been previously plated on the bottom of six-well plate B to form a feeder layer, which is labeled as pore B. In a preferred embodiment, the preparation steps of the feeder layer cells are as follows: MEF cells are inoculated into a cell culture dish. Specifically, after the cell density reaches 70% to 85%, they are treated with mitomycin C. The addition amount of mitomycin C is 10 μM, and the treatment time is 2.5 h to obtain feeder layer cells. The feeder layer cells can be cryopreserved and thawed in advance into the corresponding pore B before use. The feeder layer cells support the growth and differentiation of chinchilla iPSC. The feeder layer cells provide a microenvironment for maintaining the growth of dermal cells in pore B.

[0055] The dermal cells and feeder layer cells in pore B are cultured with Medium II for a culture time of ≥5 days, and typical IPSC-like cells can be seen to appear. Subsequently, IPSC cell clusters are formed in pore B, and the proportion gradually increases. At 18 to 20 days after transcription factor transfection, the IPSC cell clusters account for 80% to 95% of the total cells in pore B. The chinchilla dermal cells are successfully reprogrammed into IPSC, with high expression of the stemness factors of chinchilla IPSC and high cell reprogramming efficiency of chinchilla IPSC.

[0056] Example 1

[0057] The reprogramming medium of this example includes Medium I and Medium II.

[0058] Medium I contains 5 v / v% KnockOut™ Serum Replacement (KSR), 0.2 v / v% Nucleosides, 0.2 v / v% GlutaMax, 0.1 mM non-essential amino acids (NEAA), 0.02 mM 2-mercaptoethanol, 1000 U / mL leukemia inhibitory factor (LIF), 0.3 μM PD0325901, and 0.1 μM CHIR-99021, with the balance being DMEM medium.

[0059] Medium II is a complete medium for C57BL / 6 mouse embryonic stem cells, containing 5 v / v% fetal bovine serum (FBS), 0.5 v / v% Glutamine, 0.5 v / v% NEAA, 0.01 v / v% LIF, and 0.05 v / v% 2-mercaptoethanol.

[0060] The method for reprogramming chinchilla somatic cells into pluripotent stem cells in this example includes the following steps:

[0061] Inoculate chinchilla dermal cells into six-well plate A, labeled as well A. Specifically, on the day after inoculating the chinchilla dermal cells into well A, replace the dermal cell medium. The dermal cell medium contains 20 v / v% FBS and 1 v / v% penicillin / streptomycin double antibody, with the balance being high-glucose DMEM medium.

[0062] Perform reprogramming transfection on the dermal cells in well A. Use the Epi5™ Episomal iPSC Reprogramming Kit (the transcription factors include Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1, and L-Myc). Add 1 μL of each of the two tubes of plasmids, Epi5™ Reprogramming Vectors (Tube A) and Epi5™ p53&EBNA Vectors (Tube B), to well A. Then, culture the dermal cells in well A after plasmid transfection. Culture for 24 h using the dermal cell medium at a culture temperature of 37°C;

[0063] Replace the medium in well A with Medium I for culture, and change the medium every day. Culture the dermal cells in well A until the cell confluence reaches 100% in 3 days;

[0064] Passage the dermal cells in well A to six-well plate B at a ratio of 1:3. Feeder cells have been pre-coated on the bottom of six-well plate B to form a feeder layer, labeled as well B. Among them, the preparation steps of the feeder cells are as follows: Inoculate mouse embryonic fibroblast (MEF) cells into a cell culture dish. Specifically, after the cell density reaches 80%, treat with mitomycin C. The addition amount of mitomycin C is 10 μM, and the treatment time is 2.5 h to obtain feeder cells.

[0065] The dermal cells and feeder cells in well B were cultured using Medium II, and the medium was changed every 2 days. The feeder cells in well B aged and the dermal cells apoptosed. On the 10th day after transfection, observation was carried out under a microscope. Please refer to Figure 1 Figures 1a and 1b. In well B, the visible cells presented the typical shape of IPSC cells, and the proportion gradually increased. On the 19th day after transfection with transcription factors, please refer to Figure 1 Figure 1c. The cell clusters of chinchilla IPSC accounted for about 85% of the total cells in well B. The reprogramming efficiency of chinchilla IPSC was high.

[0066] Example 2

[0067] The reprogramming medium of this example includes Medium I and Medium II;

[0068] Medium I contains 30 v / v% KSR, 3 v / v% Nucleosides, 3 v / v% GlutaMax, 3 mM NEAA, 1 mM 2-mercaptoethanol, 3000 U / mL LIF, 10 μM PD0325901 and 10 μM CHIR-99021, and the balance is DMEM medium.

[0069] Medium II is the complete medium for C57BL / 6 mouse embryonic stem cells, containing 20 v / v% FBS, 3 v / v% Glutamine, 3 v / v% NEAA, 0.06 v / v% LIF, 0.3 v / v% 2-mercaptoethanol.

[0070] The method for reprogramming chinchilla somatic cells into pluripotent stem cells in this example includes the following steps:

[0071] The chinchilla dermal cells were inoculated into six-well plate A and labeled as well A. Specifically, on the day after the chinchilla dermal cells were inoculated into well A, the dermal cell medium was changed. The dermal cell medium contains 20 v / v% FBS, 1 v / v% penicillin / streptomycin double antibody, and the balance is DMEM high-glucose medium.

[0072] Reprogramming transfection is performed on the dermal cells in well A. The transcription factors include Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1, and L-Myc. For the reprogramming transfection, the Epi5™ Episomal iPSC Reprogramming Kit is used. Add 1 μL of each of the two tubes of plasmids, namely Epi5™ Reprogramming Vectors (Tube A) and Epi5™ p53&EBNA Vectors (Tube B), into well A. Then, culture the dermal cells in well A after plasmid transfection. Culture them in dermal cell medium for 24 h at a culture temperature of 37°C;

[0073] Replace the medium in well A with Medium I and culture the cells, changing the medium daily. Culture the dermal cells in well A for 3 days until the cell confluence reaches 100%;

[0074] Passage and transfer the dermal cells in well A to six-well plate B. Specifically, when the cells in well A are confluent, passage the dermal cells in well A at a ratio of 1:3 into six-well plate B, where feeder cells have been pre-plated in six-well plate B to form a feeder layer, labeled as well B. Among them, the preparation steps of the feeder cells are as follows: Inoculate MEF cells into a cell culture dish. Specifically, after the cell density reaches 80%, treat them with mitomycin C. The addition amount of mitomycin C is 10 μM, and the treatment time is 2.5 h to obtain feeder cells.

[0075] Subsequently, culture the cells in well B with Medium II and change the medium every 2 days. The feeder cells in well B age and the dermal cells apoptose. On the 9th day after transfection, observe through a microscope. Please refer to Figure 2 Figures 2a and 2b in it. In well B, the visible cells show a typical IPSC cell shape, and the proportion gradually increases. On the 18th day after plasmid transfection, please refer to Figure 2 Figure 2c in it. The cell clusters of chinchilla IPSCs account for about 90% of the total cells in well B. The cell reprogramming efficiency of chinchilla IPSCs is high.

[0076] Comparative Example 1

[0077] The difference between Comparative Example 1 and Example 1 is that the dermal cells in well A are cultured with Medium II until the cell confluence reaches 100%, which takes about 3 days. The dermal cells and feeder cells in well B are cultured with Medium I. Observe through a microscope one month after plasmid transfection. Please refer to Figure 3, typical iPSC cell morphology still did not appear in well B. Only a large number of small cells were seen in well B, which might be the precursor cells of chinchilla iPSC, and the cell reprogramming failed. In addition, medium II containing 10 μM of PD0325901 and 10 μM of CHIR-99021 was also tried to culture the cells after plasmid transfection, but typical iPSC cell morphology did not appear within one month.

[0078] Comparative Example 2

[0079] The difference between Comparative Example 2 and Example 1 was that the dermal cells in well A were cultured with medium I until the cell confluence reached 100%, and the dermal cells in well B continued to be cultured with medium I. On the 20th day after reprogramming transfection, please refer to Figure 4 , the cell clusters of chinchilla iPSC accounted for 50 - 60% of the total cells in well B, and the cell reprogramming efficiency of chinchilla iPSC was relatively low.

[0080] Comparative Example 3

[0081] The difference between Comparative Example 3 and Example 1 was that the dermal cells in well A were cultured with medium III until the cell confluence reached 100%, where medium III did not contain PD0325901 and CHIR-99021 compared with medium I.

[0082] Observed by microscope one month after plasmid transfection, please refer to Figure 5 , typical iPSC cell morphology still did not appear in well B. Only a large number of small cells were seen in well B, which might be the precursor cells of chinchilla iPSC, and the cell reprogramming failed.

[0083] Comparative Example 4

[0084] The difference between Comparative Example 4 and Example 1 was that the dermal cells in well A were cultured with medium IV until the cell confluence reached 100%, where medium IV did not contain GlutaMax compared with medium I, but added an equal amount of glutamine.

[0085] On the 20th day after plasmid transfection, please refer to Figure 6 , the cell clusters of chinchilla iPSC accounted for about 50% of the total cells in well B, and the cell reprogramming efficiency of chinchilla iPSC was relatively low.

[0086] According to the microscope observation results of Example 1, Example 2 and Comparative Examples 1 - 4, it was found that the combination of medium I and medium II could obtain high-quality chinchilla iPSC, and the cell reprogramming efficiency of chinchilla iPSC was high. Among them, from the microscope observation results of Example 1, Example 2 and Comparative Examples 1 - 2, it was known that the use order of medium I and medium II affected the cell reprogramming results of chinchilla iPSC, and the specific mechanism was unknown.

[0087] Meanwhile, from the microscope observation results of Example 1, Example 2 and Comparative Examples 3 to 4, it can be seen that GlutaMax, PD0325901 and CHIR-99021 in Medium Ⅰ play a synergistic role. Among them, Glutamine in Medium Ⅱ affects the induced culture of dermal cells in Well A and the self-characteristics of chinchilla dermal cells, making it difficult for Glutamine to remain stable during the culture process and spontaneously degrade. The ammonia produced by the degradation of Glutamine may affect the culture of chinchilla dermal cells.

[0088] Test Example

[0089] Test Example 1

[0090] Gene expression test: Collect the chinchilla IPSCs induced in Example 2 and perform RNA reverse transcription into cDNA. According to the instructions of the SYBRGreen qPCR premix kit, use the qPCR method to detect the expression of stemness-related genes of the chinchilla IPSCs prepared in Example 2. Among them, chinchilla dermal cells are used as the control group.

[0091] Table 1 Pluripotent stem cell-related genes and primers used

[0092]

[0093] Please refer to Figures 7 to 11 , compared with the control group, all the selected stemness genes were highly expressed in the chinchilla IPSCs induced in Example 2, that is, the stem cell characteristics of the chinchilla IPSCs were relatively high. The induction method of chinchilla induced pluripotent stem cells of the present invention can prepare high-quality chinchilla IPSC cell clusters.

[0094] Test Example 2

[0095] Immunofluorescence staining test: Perform immunofluorescence test on the chinchilla IPSCs induced in Example 2. The steps are as follows:

[0096] Gently wash the cells in Well B of the six-well plate 3 times with PBS, try to avoid cell detachment, and then add 1 ml of 4% paraformaldehyde cell fixative to each well and fix the cells for 0.5 h;

[0097] Discard the fixative and wash 2 times with an appropriate amount of PBS;

[0098] After discarding the PBS, add 0.5% TritonX-100, with TritonX-100 being 500 μL / well (diluted with PBS), permeabilize at room temperature for 15 min, and wash 2 times with PBS again;

[0099] Add 1 ml of 3% BSA for blocking and keep at room temperature for 1 h.

[0100] Discard the antibody blocking solution, and add 500 μl of the primary antibody solution (Oct-3 / 4). The primary antibody solution is diluted 1:50 with the antibody diluent, which is prepared by adding 3% BSA and 0.1% Triton X-100 to PBS. The cells in well B are incubated overnight in the primary antibody solution at 4 °C. After the incubation with the primary antibody is completed, wash 3 times with PBST, with each washing time being 10 min.

[0101] Discard the PBST, and add an appropriate amount of the secondary antibody solution. The secondary antibody solution is diluted 1:200 with the antibody diluent, which is prepared by mixing 3% BSA and 0.1% Triton X-100. The cells in well B are incubated in the secondary antibody solution at room temperature for 1 h.

[0102] Counterstain with 2 ng / ml DAPI for 10 min at room temperature.

[0103] Wash 4 times with PBST, 10 min each time.

[0104] Add 500 μL / well of counterstaining PBS for soaking, and observe and take pictures under a microscope.

[0105] Please refer to Figure 12 , the embryonic stem cell marker OCT3 / 4 of the chinchilla iPSC in Example 2 is highly expressed. It can be seen that the induced pluripotent stem cells obtained from the chinchilla have good pluripotent stem cell characteristics, and the reprogramming method of the present invention can prepare high-quality chinchilla iPSC cell clusters.

[0106] The above content is only an example and explanation of the structure of the present invention. Its description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these obvious replacement forms all belong to the protection scope of the present invention.

Claims

1. A method for reprogramming chinchilla somatic cells into pluripotent stem cells, characterized in that, It includes the following steps: The somatic cells of chinchilla are reprogrammed and transfected with transcription factors, and then cultured successively with Medium I and Medium II. The Medium I is DMEM medium containing 5v / v% - 20v / v% serum substitute, 0.2v / v% - 3v / v% nucleosides, 0.2v / v% - 3v / v% GlutaMax, 0.1 mM - 3 mM non-essential amino acid NEAA, 0.02 mM - 1 mM 2-mercaptoethanol, 1000 - 3000 U / mL leukemia inhibitory factor LIF, 0.3 μM - 10 μM PD0325901, and 1 μM - 10 μM CHIR-99021. The Medium II is the complete medium for C57BL / 6 mouse embryonic stem cells containing 5v / v% - 20v / v% FBS, 0.5v / v% - 3v / v% glutamine, 0.5v / v% - 3v / v% non-essential amino acid NEAA, 0.01 - 0.1v / v% leukemia inhibitory factor LIF, and 0.05v / v% - 0.3v / v% 2-mercaptoethanol. When culturing with the Medium II, the cells need to be cultured on a feeder layer. The transcription factors are Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1, and L-Myc.

2. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 1, characterized in that, When culturing with the Medium I, the culture time is 2 - 5 days.

3. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 1, characterized in that, When culturing with the Medium II, the culture time is ≥5 days.

4. The method for reprogramming a chinchilla somatic cell into a pluripotent stem cell according to claim 1, characterized in that, The somatic cells of chinchilla are cultured with the dermal cell medium and then reprogrammed and transfected with transcription factors. Among them, the dermal cell medium contains 15% - 25v / v% FBS and 0.4v / v% - 1v / v% penicillin / streptomycin double antibody, and the balance is DMEM high-glucose medium.

5. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 1, characterized in that, The serum substitute is KnockOut™ Serum Replacement.

6. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 1, characterized in that, Both the Medium I and Medium II include 0.1v / v% - 3v / v% penicillin / streptomycin double antibody.

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