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

Abstract

The embodiment of the invention relates to the technical field of cell reprogramming, and discloses a reprogramming culture medium and a method for reprogramming chinchilla somatic cells into pluripotent stem cells, the culture medium comprises a culture medium I and a culture medium II; the culture medium I is a DMEM (dulbecco's modified eagle medium) culture medium containing more than or equal to 5v / v% of a serum substitute, more than or equal to 0.2 v / v% of Nucleosides, more than or equal to 0.2 v / v% of GlutaMax, 0.1 mM to 10.0 mM of NEAA, 0.02 mM to 1mM of 2-mercaptoethanol, 1000 U / mL to 3000 U / mL of LIF, 0.3 [mu] M to 10 [mu] M of PD0325901 and 1 [mu] M to 10 [mu] M of CHIR-99021; the culture medium II is a C57BL / 6 mouse embryonic stem cell complete culture medium. The method comprises the following steps: reprogramming and transfecting transcription factors to the somatic cells of the chinchilla, and culturing by using a culture medium I and a culture medium II in sequence after reprogramming and transfecting the somatic cells of the chinchilla. By applying the technical scheme provided by the invention, the chinchilla IPSC 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 that have the potential to differentiate into multiple cell types. Because of their self-renewal and differentiation potential, they have broad application prospects in regenerative medicine, disease research, and drug screening. Among them, induced pluripotent stem cells (IPSCs) are pluripotent stem cells produced by somatic cell reprogramming. During the cell reprogramming process, differentiated somatic cells are reprogrammed under specific conditions and restored to a pluripotent state, or form an embryonic stem cell line. During cell reprogramming, induced pluripotent stem cells usually require reprogramming medium for culture induction. At present, conventional reprogramming medium mainly includes DMEM basal medium and nutrients.

[0003] At present, there are some literatures and reports on reprogramming cases of animal cells such as humans, mice, pigs, cattle, and sheep. In the reprogramming of animal cells, IPSCs can be successfully induced and cultured using conventional reprogramming culture media. Based on the reported cases of animal cell reprogramming, researchers hope to obtain chinchilla IPS by reprogramming chinchilla somatic cells. Chinchillas are small and fat, with long and fluffy hair at the end of their tails. They are chipmunks and are named for their soft and dense hair all over their bodies. Chinchilla somatic cells are reprogrammed to obtain chinchilla IPS, and then the chinchilla IPS are directed to differentiate in vitro, and finally the chinchilla follicle structure is obtained in vitro. The chinchilla hair grown from the chinchilla follicle structure can be used as green fur. The method of obtaining chinchilla hair through directed differentiation of chinchilla somatic cells through chinchilla IPS has important scientific research potential.

[0004] However, the animals that have achieved reprogrammed cells are quite different from the chinchilla species, and the species closest to the chinchilla only belongs to the same order. It is difficult to obtain chinchilla IPS by reprogramming chinchilla somatic cells using existing technology. Summary of the invention

[0005] In order to solve the above problems, the invention object of the first aspect of the present application is to provide a reprogramming medium, which can successfully obtain chinchilla IPSCs through induction culture of medium I and medium II, and is beneficial to improving the reprogramming efficiency of cells.

[0006] The second aspect of the invention of the present application aims to provide a method for reprogramming chinchilla somatic cells into pluripotent stem cells. The steps are simple, and the cells are cultured and induced in sequence using culture medium I and culture medium II, which is beneficial for obtaining high-quality chinchilla IPSCs while improving the efficiency of cell reprogramming.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the reprogramming medium of the present invention comprises medium I and medium II; The culture medium I is a DMEM culture medium containing ≥5 v / v% serum replacement, ≥0.2 v / v% Nucleosides, ≥0.2 v / v% GlutaMax (L-alanyl-L-glutamine), 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 culture medium II is a complete culture medium for C57BL / 6 mouse embryonic stem cells.

[0008] In some implementations, the complete culture medium for C57BL / 6 mouse embryonic stem cells contains 5 v / v% to 30 v / v% FBS, 0.2 v / v% to 3 v / v% Glutamine, 0.2 v / v% to 3 v / v% NEAA, 0.01 v / v% to 0.1 v / v% LIF, and 0.05 v / v% to 0.3 v / v% 2-mercaptoethanol.

[0009] In some implementations, both the culture medium I and the culture medium II include 0.1 v / v% to 3 v / v% of penicillin / streptomycin dual antibodies.

[0010] In some implementations, the serum replacement of the culture 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%.

[0011] In a second aspect, the method of reprogramming chinchilla somatic cells into pluripotent stem cells of the present invention uses the above-mentioned reprogramming medium, comprising the following steps: The chinchilla somatic cells were reprogrammed and transfected with transcription factors. After the reprogramming and transfection, the chinchilla somatic cells were cultured in culture medium Ⅰ and culture medium Ⅱ in sequence.

[0012] In some implementations, the transcription factors are Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1, and L-Myc.

[0013] In some implementations, the culture medium I is cultured for 2 to 5 days.

[0014] In some implementations, the culture medium II is cultured for ≥5 days.

[0015] In some implementations, when cultured in the medium II, the cells are cultured on a feeder layer.

[0016] In some implementations, the chinchilla somatic cells are cultured in a dermal cell culture medium and then reprogrammed and transfected with transcription factors, wherein the dermal cell culture medium contains 15v / v%~25v / v% FBS, 0.4v / v%~1v / v% penicillin / streptomycin dual antibodies, and the remainder is DMEM high-glucose culture medium.

[0017] Based on the above technical solutions, the present invention has the following technical effects: 1. The reprogramming medium provided by the present invention can reprogram chinchilla somatic cells (dermal cells) to differentiate into induced pluripotent stem cells. It requires few reagents and has low cost.

[0018] 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 transfected with reprogramming factors are reprogrammed into pluripotent stem cells by using medium I through the synergistic activation of reagents such as LIF, PD0325901 and CHIR-99021, and then the replacement of medium II can effectively promote the reprogramming of chinchilla somatic cells (dermal cells). When used with medium I and medium II, the differentiation efficiency is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a microscope image of the chinchilla IPS according to Example 1 of the present invention, with a scale bar of 500 μm.

[0020] Figure 2 This is a microscope image of the chinchilla IPS according to Example 2 of the present invention, with a scale bar of 500 μm.

[0021] Figure 3 This is a microscope image of the chinchilla IPS of Comparative Example 1 of the present invention, with a scale bar of 500 μm.

[0022] Figure 4 This is a microscope image of the chinchilla IPS of Comparative Example 2 of the present invention, with a scale bar of 500 μm.

[0023] Figure 5 This is a microscope image of the chinchilla IPS of Comparative Example 3 of the present invention, with a scale bar of 500 μm.

[0024] Figure 6 This is a microscope image of the chinchilla IPS of Comparative Example 4 of the present invention, with a scale bar of 500 μm.

[0025] Figure 7 This is a diagram showing the gene test results of Oct3 / 4 by chinchilla IPS in Example 2 of the present invention.

[0026] Figure 8 This is a diagram showing the results of the gene test of Sox2 by the chinchilla IPS according to Example 2 of the present invention.

[0027] Fig. 9 This is a diagram showing the gene test results of KIf4 by chinchilla IPS according to Example 2 of the present invention.

[0028] Fig.10 This is a diagram showing the gene test results of the chinchilla IPS on C-myc in Example 2 of the present invention.

[0029] Fig.11 This is a diagram showing the gene test results of Nanog by chinchilla IPS according to Example 2 of the present invention.

[0030] Fig.12 This is a diagram showing the immunofluorescence staining results of the chinchilla IPS according to Example 2 of the present invention. DETAILED DESCRIPTION

[0031] In order 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 thoroughly understood.

[0032] 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 application. The description provided herein is for illustrative purposes only and is not to be interpreted in a limiting sense. The inventive concepts 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, not exhaustive, and are not intended to limit the present disclosure to these specific embodiments. Moreover, it should be understood that the wording and terminology used herein are for the purpose of description and should not be considered restrictive unless otherwise stated. In addition, in the following detailed description, many specific details are listed in order to provide a more thorough understanding of the present disclosure.

[0033] However, it is obvious to those skilled in the art that the embodiments of the present disclosure can be practiced without these specific details. In other cases, features known to those skilled in the art are not described in detail to avoid unnecessary complex descriptions. It is intended that all substitutions, replacements, modifications and equivalents obvious to those skilled in the art are included within the scope of the present disclosure. According to the present disclosure, all compounds / compositions disclosed herein and their preparation methods, applications and uses can be prepared and implemented without undue experimentation.

[0034] Thus, although the compounds / compositions and methods of the present disclosure have been described according to specific embodiments, it will be apparent to those skilled in the art that changes may be made to the formulations, compounds or compositions and / or methods, as well as the steps or order of steps of the methods described herein, without departing from the spirit and scope of the inventive concepts of the present disclosure.

[0035] As used herein, any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in conjunction with the implementation is included in at least one embodiment. The phrase "in one embodiment" appearing in multiple places in the specification does not necessarily refer to the same embodiment.

[0036] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0037] The present application is described in further detail below.

[0038] The reprogramming medium of the present invention comprises medium I and medium II; Culture medium I contains ≥5 v / v% serum replacement, ≥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, and the remainder is DMEM culture medium.

[0039] As shown in some specific embodiments, the added amount of serum substitute is preferably 5v / v%~20v / v%, the added amount of Nucleosides is preferably 0.2v / v%~3v / v%, and the added amount of GlutaMax is preferably 0.2v / v%~3v / v%.

[0040] Specifically, serum replacement provides necessary nutrients for the induced culture of chinchilla somatic cells, and can support the growth and directional differentiation of chinchilla somatic cells. NEAA provides chinchilla somatic cells with non-essential amino acids required for growth, and can also balance the pH value of the growth environment of chinchilla dermal cells, ensuring that reagents such as PD0325901 and CHIR-99021 work synergistically to promote the initiation of cell reprogramming.

[0041] Medium II was purchased from HyCyte TM C57BL / 6 mouse embryonic stem cell complete medium. The concentration of the supplements provided can be adjusted as needed.

[0042] In a preferred embodiment, the complete culture medium for C57BL / 6 mouse embryonic stem cells contains 5 v / v% to 30 v / v% FBS, 0.5 v / v% to 3 v / v% Glutamine, 0.5 v / v% to 3 v / v% NEAA, 0.01 v / v% to 0.1 v / v% LIF, and 0.05 v / v% to 0.3 v / v% 2-mercaptoethanol.

[0043] Specifically, Culture Medium II can promote the efficiency of chinchilla reprogramming, allowing more than 80% of chinchilla somatic cells (dermal cells) to be reprogrammed into induced pluripotent stem cells within a period of 18-20 days.

[0044] In a preferred embodiment, both culture medium I and culture medium II contain 0.1 v / v% to 3 v / v% of penicillin / streptomycin dual antibodies, which effectively protect chinchilla somatic cells from microbial invasion.

[0045] The method for reprogramming chinchilla somatic cells into pluripotent stem cells of the present invention uses the above-mentioned reprogramming medium, and comprises the following steps: Chinchilla somatic cells are reprogrammed and transfected with transcription factors. Specifically, chinchilla somatic cells can be preferably chinchilla dermal cells. Before reprogramming transfection, chinchilla dermal cells are inoculated into six-well plate A, marked as well A. The day after the chinchilla dermal cells are inoculated into well A, the dermal cell culture medium is replaced. The dermal cell culture medium contains 15v / v%~25v / v% FBS, 0.4v / v%~1v / v% penicillin / streptomycin dual antibodies, and the remainder is DMEM high-glucose culture medium.

[0046] The dermal cells in well A are transfected with transcription factors for reprogramming. In some specific embodiments, the transcription factors include Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1 and L-Myc. For example, the transcription factor transfection can use the Epi5™ Episomal iPSC reprogramming kit, 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, and then culture the dermal cells in well A after transcription factor transfection. In a preferred embodiment, the dermal cells in well A can be cultured in an incubator for 18 h to 24 h, specifically, for 18 h, 20 h or 24 h, and the culture temperature is 37° C. The dermal cells in well A are cultured with medium I for 2 to 5 days, with the medium changed every day. The dermal cells in well A are cultured until the cell confluence reaches 100%. Medium I promotes the rapid programming of the dermal cells in well A and reduces the apoptosis of the dermal cells in well A while programming the cells; The dermal cells in well A are transferred to six-well plate B by passage. Specifically, when the cells in well A are full, the dermal cells in well A are passaged to six-well plate B at a ratio of 1:3. The bottom of six-well plate B has been pre-painted with feeder layer cells to form a feeder layer, which is marked as well B. In a preferred embodiment, the preparation step of the feeder layer cells is to inoculate MEF cells into a cell culture dish. Specifically, after the cell density reaches 70% to 85%, the cells are treated with mitomycin C. The amount of mitomycin C added is 10 μM, and the treatment time is 2.5 hours to obtain feeder layer cells. The feeder layer cells can be frozen and revived in the corresponding well B before use. Feeder layer cells support the growth and differentiation of chinchilla IPSCs. Feeder layer cells provide a microenvironment for the dermal cells in well B to maintain cell growth.

[0047] The dermal cells and feeder layer cells in well B were cultured with medium II for ≥5 days, and typical IPSC morphology cells appeared. Subsequently, IPSC cell clusters formed in well B, and the proportion gradually increased. At 18-20 days after transcription factor transfection, IPSC cell clusters accounted for 80%-95% of the total cells in well B. The chinchilla dermal cells were successfully programmed into IPSCs, the stemness factors of chinchilla IPSCs were highly expressed, and the cell reprogramming efficiency of chinchilla IPSCs was high.

[0048] Example 1 The reprogramming medium of this embodiment includes medium I and medium II; Culture medium I contains 5 v / v% serum replacement (KSR, KnockOut™ Serum Replacement), 0.2 v / v% Nucleosides, 0.2 v / v% GlutaMax, 0.1 mM NEAA, 0.02 mM 2-mercaptoethanol, 1000 U / mL LIF, 0.3 μM PD0325901 and 0.1 μM CHIR-99021, and the remainder is DMEM culture medium.

[0049] Culture medium II is a complete culture medium for C57BL / 6 mouse embryonic stem cells, containing 5v / v% FBS, 0.5v / v% Glutamine, 0.5v / v% NEAA, 0.01v / v% LIF, and 0.05v / v% 2-mercaptoethanol.

[0050] The method of reprogramming chinchilla somatic cells into pluripotent stem cells in this embodiment comprises the following steps: The chinchilla dermal cells were inoculated into six-well plate A, marked as well A. Specifically, the day after the chinchilla dermal cells were inoculated into well A, the dermal cell culture medium was replaced, and the dermal cell culture medium contained 20 v / v% FBS, 1 v / v% penicillin / streptomycin dual antibody, and the remainder was DMEM high-glucose culture medium.

[0051] The dermal cells in well A were reprogrammed and transfected using the Epi5™ Episomal iPSC Reprogramming Kit (transcription factors include Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1 and L-Myc). 1 μL of each of the two tubes of plasmids Epi5™ Reprogramming Vectors (Tube A) and Epi5™ p53&EBNA Vectors (Tube B) was added to well A. The dermal cells in well A after plasmid transfection were then cultured in dermal cell culture medium for 24 hours at 37°C. The culture medium in well A was replaced with culture medium I, and the medium was changed every day. The dermal cells in well A were cultured for 3 days until the cell confluence reached 100%; The dermal cells in well A were passaged to six-well plate B at a ratio of 1:3. Feeder layer cells were pre-laid on the bottom of six-well plate B to form a feeder layer, which was marked as well B. The preparation step of feeder layer cells is to inoculate MEF cells into a cell culture dish. Specifically, after the cell density reaches 80%, mitomycin C is used for treatment. The amount of mitomycin C added is 10 μM, and the treatment time is 2.5 h to obtain feeder layer cells.

[0052] The dermal cells and feeder layer cells in well B were cultured with medium II, and the medium was changed every 2 days. The feeder layer cells in well B were aged and the dermal cells were apoptotic. The results were observed under a microscope 10 days after transfection. Figure 1 In 1a and 1b, cells in well B showed typical IPSC cell shape, and the proportion gradually increased. On the 19th day after transcription factor transfection, see Figure 1 In well 1c, the cell cluster of chinchilla IPSCs accounts for about 85% of the total number of cells in well B, and the cell reprogramming efficiency of chinchilla IPSCs is high.

[0053] Example 2 The reprogramming medium of this embodiment includes medium I and medium II; Culture medium I contained 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 remainder was DMEM culture medium.

[0054] Medium II is a complete medium for C57BL / 6 mouse embryonic stem cells, containing 20v / v% FBS, 3v / v% Glutamine, 3v / v% NEAA, 0.06v / v% LIF, and 0.3v / v% 2-mercaptoethanol.

[0055] The method of reprogramming chinchilla somatic cells into pluripotent stem cells of this embodiment comprises the following steps: The chinchilla dermal cells were inoculated into six-well plate A, marked as well A. Specifically, the day after the chinchilla dermal cells were inoculated into well A, the dermal cell culture medium was replaced, and the dermal cell culture medium contained 20 v / v% FBS, 1 v / v% penicillin / streptomycin dual antibody, and the remainder was DMEM high-glucose culture medium.

[0056] The dermal cells in well A were reprogrammed and transfected with transcription factors including Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1 and L-Myc. The Epi5™ Episomal iPSC Reprogramming Kit was used for reprogramming. 1 μL of each of the two tubes of plasmids Epi5™ Reprogramming Vectors (Tube A) and Epi5™ p53&EBNA Vectors (Tube B) was added to well A. The dermal cells in well A after plasmid transfection were then cultured in dermal cell culture medium for 24 hours at 37°C. The medium in well A was replaced with medium I, and the cells were cultured, with the medium changed every day. The dermal cells in well A were cultured for 3 days until the cell confluence reached 100%; The dermal cells in well A were subcultured and transferred to six-well plate B. Specifically, when the cells in well A were fully grown, the dermal cells in well A were subcultured at a ratio of 1:3 to six-well plate B. Feeder layer cells had been pre-laid in six-well plate B to form a feeder layer, which was marked as well B. The preparation step of the feeder layer cells was to inoculate MEF cells into a cell culture dish. Specifically, after the cell density reached 80%, the cells were treated with mitomycin C. The amount of mitomycin C added was 10 μM, and the treatment time was 2.5 h to obtain feeder layer cells.

[0057] Subsequently, the cells in well B were cultured with medium II, and the medium was changed every 2 days. The feeder layer cells in well B were aged and the dermal cells were apoptotic. The results were observed under a microscope 9 days after transfection. Figure 2 In 2a and 2b, cells in well B showed typical IPSC cell shape, and the proportion gradually increased. On the 18th day after plasmid transfection, see Figure 2 In well 2c, the cell cluster of chinchilla IPSCs accounts for about 90% of the total number of cells in well B, and the cell reprogramming efficiency of chinchilla IPSCs is high.

[0058] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the dermal cells in well A were cultured with medium II until the cell confluence reached 100%, which took about 3 days, while the dermal cells and feeder layer cells in well B were cultured with medium I. One month after plasmid transfection, the dermal cells and feeder layer cells were observed under a microscope. Figure 3 , the typical iPSC cell morphology still did not appear in well B, and only a large number of small cells appeared in well B, which may be chinchilla IPSC precursor cells, and cell reprogramming failed. In addition, we also tried to add medium II containing 10μM PD0325901 and 10μM CHIR-99021 to culture the plasmid-transfected cells, but the typical iPSC cell morphology did not appear within one month.

[0059] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the dermal cells in well A are cultured with medium I until the cell confluence reaches 100%, and the dermal cells in well B continue to be cultured with medium I. On the 20th day after reprogramming transfection, see Figure 4 , the cell clusters of chinchilla IPSCs accounted for 50-60% of the total cells in well B, and the cell reprogramming efficiency of chinchilla IPSCs was low.

[0060] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the dermal cells in well A are cultured using culture medium III until the cell confluence is 100%, wherein culture medium III is not supplemented with PD0325901 and CHIR-99021 relative to culture medium I.

[0061] One month after plasmid transfection, observe by microscope, see Figure 5 , the typical iPSC cell morphology still did not appear in well B. Only a large number of small cells appeared in well B, which might be chinchilla IPSC precursor cells, and cell reprogramming failed.

[0062] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the dermal cells in well A are cultured using medium IV until the cell confluence is 100%, wherein medium IV is not supplemented with GlutaMax but supplemented with an equal amount of glutamine relative to medium I.

[0063] On day 20 after plasmid transfection, see Figure 6 , the cell cluster of chinchilla IPSCs accounted for about 50% of the total cells in well B, and the cell reprogramming efficiency of chinchilla IPSCs was low.

[0064] According to the microscopic observation results of Example 1, Example 2 and Comparative Examples 1 to 4, it can be seen that the combination of medium I and medium II can obtain high-quality chinchilla IPSCs, and the cell reprogramming efficiency of chinchilla IPSCs is high. Among them, according to the microscopic observation results of Example 1, Example 2 and Comparative Examples 1 to 2, the order of using medium I and medium II affects the cell reprogramming results of chinchilla IPSCs, and the specific mechanism is unknown.

[0065] At the same time, it can be seen from the microscopic observation results of Example 1, Example 2 and Comparative Examples 3-4 that GlutaMax, PD0325901 and CHIR-99021 in culture medium I act synergistically. Among them, Glutamine in culture medium II affects the induced culture of dermal cells in well A. The inherent characteristics of chinchilla dermal cells make 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.

[0066] Test example Test Example 1 Gene expression test: The chinchilla IPSCs induced in Example 2 were collected, and RNA was reverse transcribed into cDNA. According to the instructions of the SYBRGreen qPCR premix kit, the expression of stemness-related genes in the chinchilla IPSCs prepared in Example 2 was detected using the qPCR method, wherein chinchilla dermal cells were used as the control group.

[0067] Table 1 Pluripotent stem cell related genes and primers used

[0068] See also Figure 7~Figure 11 Compared with the control group, the chinchilla IPSCs induced in Example 2 detected that all the selected stemness genes were highly expressed, that is, the chinchilla IPSCs had high stem cell characteristics. The induction method of the chinchilla induced pluripotent stem cells of the present invention can prepare high-quality chinchilla IPSC cell clusters.

[0069] Test Example 2 Immunofluorescence staining test: The chinchilla IPSC induced in Example 2 was subjected to immunofluorescence staining test, and the steps were as follows: The cells in the six-well plate B were gently washed three times with PBS to avoid cell shedding as much as possible, and then 1 ml of 4% paraformaldehyde cell fixative was added to each well and the cells were fixed for 0.5 h; Discard the fixative and take an appropriate amount of PBS to wash twice; After discarding PBS, add 0.5% TritonX-100, TritonX-100 at 500 uL / well (diluted with PBS), permeabilize at room temperature for 15 min, and wash twice with PBS again; Add 1 ml of 3% BSA for blocking and keep at room temperature for 1 h.

[0070] Discard the antibody blocking solution, add 500ul of primary antibody solution (Oct-3 / 4), the primary antibody solution is diluted 1:50 with antibody diluent, the antibody diluent is prepared by adding 3% BSA and 0.1% TritonX-100 to PBS, and the cells in well B are incubated in the primary antibody solution overnight at 4°C. After the primary antibody incubation is completed, wash 3 times with PBST, each washing time is 10 minutes.

[0071] PBST was discarded, and an appropriate amount of secondary antibody solution was added. The secondary antibody solution was diluted at 1:200 with antibody diluent, which was prepared by mixing 3% BSA and 0.1% TritonX-100. The cells in well B were incubated in the secondary antibody solution at room temperature for 1 h.

[0072] 2 ng / ml DAPI was used for counterstaining, the counterstaining time was 10 min, and the counterstaining temperature was room temperature.

[0073] Wash 4 times with PBST, 10 min each time.

[0074] Add 500 uL / well counterstaining PBS and soak, then observe and take pictures under a microscope.

[0075] See also Fig.12The embryonic stem cell marker OCT3 / 4 of the chinchilla IPSC in Example 2 is highly expressed, indicating 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.

[0076] The above contents are merely examples and illustrations of the structure of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.

Claims

1. A reprogramming medium, characterized in that It includes culture medium I and culture medium II; The culture medium I is a DMEM culture medium containing ≥5 v / v% serum replacement, ≥0.2 v / v% Nucleosides, ≥0.2 v / v% GlutaMax, 0.1 mM to 10 mM NEAA, 0.02 mM to 1 mM 2-mercaptoethanol, 1000 to 3000 U / mL LIF, 0.3 μM to 10 μM PD0325901, and 1 μM to 10 μM CHIR-99021; The culture medium II is a complete culture medium for C57BL / 6 mouse embryonic stem cells.

2. The reprogramming medium according to claim 1, characterized in that The complete culture medium for C57BL / 6 mouse embryonic stem cells contains 5v / v%~30v / v% FBS, 0.5v / v%~3v / v% Glutamine, 0.5v / v%~3v / v% NEAA, 0.01v / v%~0.1v / v% LIF, and 0.05v / v%~0.3v / v% 2-mercaptoethanol.

3. The reprogramming medium according to claim 1 or 2, characterized in that The culture medium I and culture medium II both include 0.1 v / v% to 3 v / v% of penicillin / streptomycin dual antibodies.

4. The reprogramming medium according to claim 1, characterized in that The serum replacement of the culture 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%.

5. A method for reprogramming chinchilla somatic cells into pluripotent stem cells, characterized in that: Using the reprogramming medium according to any one of claims 1 to 4, comprising the following steps: The chinchilla somatic cells were reprogrammed and transfected with transcription factors. After the reprogramming and transfection, the chinchilla somatic cells were cultured in culture medium Ⅰ and culture medium Ⅱ in sequence.

6. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 5, characterized in that: The transcription factors are Oct4, Sox2, Lin28, Klf4, mp53DD, EBNA1 and L-Myc.

7. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 5, characterized in that: The culture medium I is cultured for 2 to 5 days.

8. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 5, characterized in that: The culture medium II is cultured for a period of ≥5 days.

9. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 5, characterized in that: When cultured in the medium II, the cells are cultured on a feeder layer.

10. The method for reprogramming chinchilla somatic cells into pluripotent stem cells according to claim 5, characterized in that: The chinchilla somatic cells are cultured in a dermal cell culture medium and then reprogrammed and transfected with transcription factors, wherein the dermal cell culture medium contains 15% to 25 v / v% FBS, 0.4 v / v% to 1 v / v% penicillin / streptomycin dual antibodies, and the remainder is DMEM high-glucose culture medium.

Images