CN108060122B - Small molecule compound combination and method for preparing chondrocytes by using cells induced to differentiate by small molecule compound combination - Google Patents

Small molecule compound combination and method for preparing chondrocytes by using cells induced to differentiate by small molecule compound combination Download PDF

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CN108060122B
CN108060122B CN201610975996.7A CN201610975996A CN108060122B CN 108060122 B CN108060122 B CN 108060122B CN 201610975996 A CN201610975996 A CN 201610975996A CN 108060122 B CN108060122 B CN 108060122B
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repsox
vpa
chir99021
forskolin
epz004777
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胡敏
李燕皎
王兆杰
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Shenzhen Zhende Keats pharmaceutical research and Development Co.,Ltd.
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Abstract

The invention discloses a small molecular compound combination and a method for preparing chondrocytes by using cells induced and differentiated by the small molecular compound combination. The method carries out directional induction on differentiated cells to prepare cartilage cells, wherein the directional induction comprises the steps of inhibiting the activity of lysine deacetylase, inhibiting a TGF-beta signal pathway, inhibiting the activity of PKC, inhibiting the activity of DNA methyltransferase and inhibiting the activity of histone methyltransferase; the small molecule compound combination comprises compounds for regulating and controlling the signal path and/or enzyme activity. The invention can transdifferentiate differentiated cells into chondrocytes by staged induction of small molecular compounds, can realize accurate quality control in each step, and is convenient for standardized operation and large-scale production. The method provided by the invention has wide donor source, and the patient can be used as the donor to obtain the chondrocyte required by basic research, clinical treatment or tissue engineering production in a short time.

Description

Small molecule compound combination and method for preparing chondrocytes by using cells induced to differentiate by small molecule compound combination
Technical Field
The invention relates to the fields of cell biology, tissue engineering and regenerative medicine, in particular to a small molecular compound combination and a method for preparing chondrocytes by using cells induced and differentiated by the small molecular compound combination.
Background
Articular cartilage covers the ends of the bone and provides shock absorption and lubrication to the glenoid fossa. Cartilage has a poor intrinsic healing capacity as an avascular, nonlymphoid and denervated tissue. Cartilage damage has caused major economic and social problems worldwide due to various diseases such as Osteoarthritis (OA) caused by trauma or cartilage defects that increase with age, and has now become the fourth leading cause of disability. It has been reported that about 9% of OA patients in the hip or knee joints in the united states aged 30 and older have annual medical costs of $ 286 million. Therefore, the safe and effective cartilage repair technology or material industry has great value. Autologous chondrocyte transplantation is a current method for repairing focal damage of articular cartilage, but the method causes new cartilage defect due to the need of tissue biopsy and collection of healthy chondrocytes. In addition, since the in vitro expansion of chondrocytes causes cell dedifferentiation, chondrocytes tend to form fibrotic cells, thereby forming fibrous cartilage tissue containing type I collagen, which is not conducive to repair of defective cartilage lesions. In recent years, research on the repair of cartilage damage using stem cells has been increasing, and currently, MSC has been identified as the most suitable cell for cartilage damage repair. However, MSCs have limitations such as a decrease in proliferation potency with the age of a patient, a limited number of cells obtained from a patient tissue, and the like, and thus have limited clinical applications. The theory of obtaining autologous hyaline chondrocytes by inducing pluripotent stem cells (ipscs) is feasible, but the safety problems of introducing foreign genes and forming teratoma risks in the technology are not solved at present.
A variety of differentiated cells such as skin fibroblasts have the advantages of abundant sources, easy acquisition and easy long-term mass amplification culture in vitro. At present, differentiated cells such as skin fibroblasts can be directly induced into functional cells of various types such as myoblasts, neurons, hepatocytes and osteoblasts by utilizing a cell transdifferentiation technology; or the pluripotent stem cells are induced firstly and then are further induced into corresponding functional cells in a targeted manner. The functional cells obtained by directly or indirectly inducing a specific differentiated cell such as a skin fibroblast by using the cell transdifferentiation technique as described above no longer maintain the molecular characteristics and functions of the source cell, but obtain the molecular characteristics and cellular functions typical of the corresponding target cell. Currently, the above-mentioned induced functional cells have been gradually applied to disease model research, clinical treatment research and tissue engineering research.
Traditional cellular transdifferentiation needs to be achieved by the introduction of specific exogenous genes, sometimes with the synergistic effect of corresponding small molecule compounds, cytokines or recombinant proteins. The literature has reported that a certain differentiated cell is induced into another functionally differentiated cell by using a specific exogenous gene. For example, the reports of transdifferentiation of dermal fibroblasts into osteoblasts having bone forming effect in vitro and in vivo by using BMP-2, BMP-7 and LMP3 alone or in synergistic effect have been reported in the literature. However, the introduction of exogenous genes has the risk of causing tumors, and may cause target cells to generate immunogenicity, so that the popularization and the application are difficult. In 2013, dunkokui reports that the transdifferentiation process of reprogramming mouse skin fibroblasts into nerve cells can be realized only by using small molecular compounds or combinations thereof, and confirms that the cell transdifferentiation technology has the advantages of short induction process, stable induction system, easiness in quality control, low cost, no tumorigenic risk caused by exogenous gene insertion, good safety and stability of obtained target cells, no immunogenicity, and potential clinical application value and industrial prospect. Thereafter, chinese patent application No. 201410075246.5 provides a method for inducing transdifferentiation of differentiated cells into neural stem cells and uses thereof. Specifically, the application relates to the use of a combination of Histone Deacetylase (HDACs) inhibitors, glycogen synthase kinase (GSK-3) inhibitors and transforming growth factor beta (TGF-beta) signaling pathway inhibitors to induce transdifferentiation of differentiated cells such as fibroblasts and epithelial cells into neural stem cells with good pluripotency and passaging stability under normal physiological hypoxic environment. The Chinese patent application with the application number of 201610213644.8 provides an induction medium for inducing the transdifferentiation of fibroblasts into cardiomyocytes, a method and application thereof, wherein the induction medium comprises a basic medium and an induction small molecule combination, the induction small molecule combination is 6TCFOW or SCFOV, wherein 6 is E61541, T is phenylpropylamine, C is CHIR99021, F is forskolin, O is Dorsomorphin, W is IWR-1, S is SB431542, and V is valproic acid. The application can induce the transdifferentiation of fibroblasts into cardiomyocytes by using the induction culture medium. Currently, the results of obtaining schwann cells (THOMA EC, et al, 2014), nerve cells (HU W, et al, 2015), islet cells (Sheng Ding, et al, 2015) by using simple small molecule compounds or their combinations to induce human differentiated cells such as skin fibroblasts have been reported successively.
Because human and mouse have about 25% of gene difference, the feasibility of applying the technical scheme of the patent application successfully obtained in the mouse cell experiment to the transdifferentiation of human homogeneous cells is not high; on the other hand, since the specific theoretical basis and technical means for obtaining different target cells by inducing the transdifferentiation of the same cell type are different, the applicant has repeatedly tested the above reported technical schemes, and has failed to successfully apply the transdifferentiation technical scheme applied to the mouse cell to the transdifferentiation of the same cell type of the human, or to induce the transdifferentiation of the human differentiated cell into the chondrocyte.
Disclosure of Invention
The invention provides a small molecular compound combination and a method for preparing chondrocytes by using cells induced to differentiate by the small molecular compound combination. The invention uses small molecule compound combination to process differentiated cells in stages, and can rapidly, stably and orderly prepare a large amount of chondrocytes or products thereof.
In a first aspect of the present invention, there is provided a method for preparing chondrocytes by inducing differentiated cells, wherein the method comprises performing directional induction on the differentiated cells to prepare chondrocytes, wherein the directional induction comprises inhibiting the activity of lysine deacetylase, inhibiting the signaling pathway of TGF- β (preferably, the signaling pathway involved in TGF- β type I receptor), inhibiting the activity of PKC, inhibiting the activity of DNA Methyltransferase (DNMT), and inhibiting the activity of Histone Methyltransferase (HMT); activates the signaling pathway of WNT/beta-catenin, activates the signaling pathway of cAMP (preferably EPAC/RAP1), and activates the signaling pathway of RA.
Further, the directional induction also comprises the steps of inhibiting the activity of DNA Methyltransferase (DNMT) and/or inhibiting the activity of Histone Methyltransferase (HMT), inhibiting a signal path of JNK and/or inhibiting a signal path of ROCK and/or activating a signal path of smad4/smad5 and/or inhibiting the preparation of Histone demethylase to obtain the chondrocyte.
Because the directional induction treatment has great harm to cells and influences the induction rate, the pretreatment is further carried out before the directional induction, and the pretreatment comprises the steps of inhibiting a TGF-beta signal pathway, activating a WNT/beta-catenin signal pathway and activating a cAMP signal pathway; the pretreatment comprises the inhibition of the activity of lysine deacetylase, the inhibition of the TGF-beta signaling pathway, the activation of the WNT/beta-catenin signaling pathway and the activation of the cAMP signaling pathway. The pretreatment process serves to activate the differentiated cells, thereby increasing the survival rate of the cells during the directed induction phase.
Preferably, the method comprises pretreatment, first-stage directional induction and second-stage directional induction, wherein the pretreatment comprises the steps of inhibiting a signaling pathway of a TGF-beta receptor, activating a signaling pathway of WNT/beta-catenin and activating a signaling pathway of cAMP;
or the pre-treatment comprises inhibiting the activity of lysine deacetylase, inhibiting a signaling pathway of TGF-beta receptor, a signaling pathway of WNT/beta-catenin and a signaling pathway of activating cAMP;
the first-stage directional induction and the second-stage directional induction respectively comprise the steps of inhibiting the activity of lysine deacetylase, inhibiting a signaling pathway of a TGF-beta receptor, inhibiting the activity of PKC, activating a signaling pathway of WNT/beta-catenin, activating a signaling pathway of cAMP and activating a signaling pathway of RA;
or the first stage directional induction and the second stage directional induction comprise the activity of lysine deacetylase, the signal pathway of TGF-beta receptor is inhibited, the activity of PKC is inhibited, the signal pathway of WNT/beta-catenin is activated, the signal pathway of cAMP is activated, the signal pathway of RA is activated, the activity of DNA Methyltransferase (DNMT) is inhibited, the activity of Histone Methyltransferase (HMT) is inhibited, the signal pathway of JNK is inhibited, the signal pathway of ROCK is inhibited, the signal pathway of smad4/smad5 is activated, and/or the preparation of the chondrocyte is inhibited.
In some embodiments of the invention, the differentiated cell is derived from a mammal, such as a human, wherein the differentiated cell comprises a fibroblast, an epithelial cell, an adipocyte or a blood cell. Preferably the differentiated cell is a fibroblast.
In a second aspect of the invention, there is also provided a combination of small molecule compounds comprising a compound for modulating (activating or inhibiting) or chronologically staging the signaling pathway and/or enzyme activity as described above.
The small molecule compound combination comprises an inhibitor or an activator of signal-to-channel and an inhibitor or an activator of enzyme.
The small molecule compound combination comprises the following components: lysine deacetylase inhibitors, TGF-beta receptor inhibitors, PKC inhibitors, WNT/beta-catenin agonists, cAMP agonists, RAR agonists, DNMT inhibitors, and HMT inhibitors; on the basis of this combination, the present invention also provides a preferred combination of small molecule compounds further comprising at least one of ascorbate (ascorbic acid), a histone demethylase inhibitor, an inhibitor of JNK, an inhibitor of ROCK (Rho-associated protein kinase) and an activator of smad4/smad 5.
The lysine Deacetylase Inhibitor comprises sodium phenacylbutyrate, butyrate, sodium butyrate, MC1568, CI994 (tacedinine), Chidamide, CAY10683 (Santacacruzamate A), CUDC-907, M344(Histone Deacetylase Inhibitor III), LAQ824(NVP-LAQ824, Dacinostat), Pracinostat (SB939), VPA, Scriptaid, Apicidin, LBH-589 (Panobiostat), MS-275, SAHA (Vorinostat), Trichostatin (TSA), Psampalamaratin A, PCI-24781(Abexinostat), Rocilinostat (ACY-1215), cececetostat (PXD 0103), 4-Phenylbutyrate (4-4), PHaxolide B-3547, MGC-7, BCG-7, CAPB-7, BCG-7, BCA-BTX-B-7, BCA, BCG-B-7, BCG-7, BCA-S-C-7, BCA, BCG-V-B-P, BCA, BCG-P-103, BCG-B-P, BCG-7, BCG-B-7, BCG-B, BCG-7, BCG-B, BCG-B, BCG-7, BCG-B, BCA, BCG-B, BCG-7, BCG-B, BCG-B, BCG;
the inhibitor of the TGF-beta signaling pathway includes at least one of 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Gallisiertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476, and SB 505124;
the PKC inhibitor comprises at least one of Go6983, Ro31-8220 Mesylate, Go6976 and Bisindolylmaleimide I (GF 109203X);
the WNT/beta-catenin signal channel activator comprises MAY-262611, CHIR98014, CHIR99021, LiCl and Li2CO3At least one of TD114-2, AZD2858, AZD1080, BIO, Kenpaulolone, TWS119, LY2090314, CBM1078, SB216763 and AR-A014418;
the cAMP activator comprises at least one of EPAC/RAP1 activator, 8-Bromo-cAMP, dibutyl-cAMP and Sp-8-Br-cAMPs;
the activator of the EPAC/RAP1 signaling pathway comprises at least one of Forskolin, IBMX, Prostaglandin E2(PGE2), NKH477, 8-pCPT-2' -O-Me-cAMP, GSK256066, Apreminiramate (CC-10004), Roflumilast, Cilomilast, Rolipram and Millinone;
the activator of the RA signaling pathway comprises at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, Vitamin A, Vitamin A derivatives, and 13-cis RA;
the inhibitor of the ROCK signal pathway comprises at least one of Y-27632, Y-276322 HCl, Thiazovivin, Ripasudil (K-115), Fasuudil (HA-1077) HCl, GSK429286A, RKI-1447, and PKI-1313;
the Inhibitor of the JNK signaling pathway comprises at least one of SP600125, JNK Inhibitor IX, AS601245, AS602801 and JNK-IN-8;
the DNMT inhibitor comprises at least one of RG108, Thioguanine, 5-Aza-2' -deoxycytidine (Decitabine), SGI-1027, Zebularine, and 5-Azacytidine (AZA);
the HMT inhibitor comprises at least one of EPZ004777, EPZ5676, GSK503, BIX 01294 and SGC 0946;
the inhibitor of histone demethylase activity comprises at least one of parnate (tranylcyclopromine), tranylcyclopromine (2-PCPA) HCl, SP2509, 4SC-202, ORY-1001(RG-6016), GSKJ1 and GSK-LSD 1;
the activator of the smad4/smad5 signal channel is Kartogenin.
Further, the small molecule compound combination adopts any one of the following combinations:
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125+AM580；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+5-Aza-2'-deoxycytidine+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125+AM580+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+SP600125+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine+ascorbate+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+ascorbate+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate+5-Aza-2'-deoxycytidine+Parnate。
the small molecule compound combination comprises a pretreatment compound for treating differentiated cells according to time sequence and staged treatment, a first stage directed induction compound and a second stage directed induction compound;
preferably, the pretreatment compound employs any one of the following combinations:
CHIR99021+Repsox；
BIO+Repsox；
BIO+SB431542；
CHIR99021+SB431542；
BIO+SB431542；
VPA+SB431542；
VPA+Repsox；
VPA+CHIR99021+Repsox；
VPA+CHIR99021+SB431542；
VPA+BIO+Repsox；
VPA+BIO+SB431542；
CHIR99021+Repsox+Forskolin；
CHIR99021+Repsox+Rolipram；
BIO+Repsox+Rolipram；
BIO+SB431542+Rolipram；
BIO+SB431542+Forskolin；
BIO+Repsox+Forskolin；
CHIR99021+SB431542+Rolipram；
CHIR99021+SB431542+Forskolin；
VPA+CHIR99021+Repsox+Forskolin；
VPA+BIO+Repsox+Forskolin；
VPA+CHIR99021+Repsox+Rolipram；
VPA+CHIR99021+SB431542+Forskolin；
VPA+SB431542+Rolipram；
VPA+Repsox+Rolipram；
VPA+SB431542+Forskolin；
VPA+Repsox+Forskolin；
CHIR99021+Repsox+Forskolin+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Parnate；
VPA+CHIR99021+Repsox+Parnate；
CHIR99021+Repsox+Parnate；
BIO+Repsox+Parnate；
BIO+SB431542+Parnate；
CHIR99021+SB431542+Parnate；
VPA+SB431542+Parnate；
VPA+Repsox+Parnate；
VPA+Repsox+Forskolin+Parnate；
CHIR99021+Repsox+Parnate；
VPA+Repsox+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Parnate；
preferably, the first stage directional inducing compound employs any one of the following combinations:
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125+AM580；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+5-Aza-2'-deoxycytidine+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125+AM580+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+SP600125+Parnate
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine+ascorbate+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+ascorbate+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate+Parnate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate+5-Aza-2'-deoxycytidine+Parnate；
preferably, the second stage directional inducing compound employs any one of the following combinations:
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+5-Aza-2'-deoxycytidine+SP600125+AM580；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+SP600125+Y-27632+5-Aza-2'-deoxycytidine+ascorbate；
VPA+CHIR99021+Repsox+Forskolin+Go6983+TTNPB+EPZ004777+AM580+Y-27632+ascorbate+5-Aza-2'-deoxycytidine。
in some embodiments of the present invention, effective concentrations of specific small molecule compounds are as follows, and the concentration ranges given below are merely for reference and can be adapted accordingly, and the concentrations can be adapted if other small molecules are substituted for the following small molecules.
The concentration of Forskolin is 2-20 MuM; the Repsox concentration is 2-15 uM; CHIR99021 concentration is 1-10 μ M; VPA concentration is 0.5 mM-1.5 mM; TTNPB concentration is 3-8 μ M; the concentration of AM580 is 0.03-0.08 mu M; the concentration of EPZ004777 is 3-8 mu M; the concentration of Go6983 is 1-15 mu M; the concentration of Y-27632 is 3-15 mu M, and the concentration of L-ascorbacid 2-phosphate is 0.15-0.25 mM; the concentration of the SP600125 is 1-15 mu M; the concentration of the 5-Aza-2' -deoxycytidine is 0.5-15 mu M.
Other cell types, such as osteoblasts, may also be prepared using the methods of the invention or by adapting cells induced to differentiate on the basis of the methods of the invention. If the small molecule compound combination provided by the invention is used for preparing other cells except chondrocytes and osteoblasts, the concentration of the corresponding small molecule compound can be adjusted according to actual needs, and the adjustment can be made on the small molecule compound combination.
The second phase directional inducing compound also comprises cell growth factor BMP4 and/or PDGF-AB and/or basic fibroblast growth factor b-FGF protein, and regulates the growth of cells.
In a third aspect of the invention, a kit or culture medium comprising the small molecule compound combination is also provided.
In the fourth aspect of the invention, the small molecule compound combination, a kit containing the small molecule compound combination or the application of a culture solution/medium are also provided, and the small molecule compound combination is used for preparing cartilage cells or regenerative medicine seed cells, tissue engineering seed cells and products thereof which take the cartilage cells as source cells in an in vivo mobilization induction or in vitro induction; can be used for basic research, clinical treatment and research, development and production of tissue engineering products. Can be used for basic research, clinical treatment and research, development and production of tissue engineering products.
In the fifth aspect of the invention, the chondrocyte prepared by the method or prepared by using the small molecule compound combination is also provided. The chondrocytes have the characteristics of standard chondrocytes, belong to hyaline cartilage, and are rarely subjected to chondrocyte hypertrophy in vitro culture; high purity, mass production and good industrialization prospect.
In the sixth aspect of the invention, the application of the chondrocyte and the product thereof is also provided, such as the small molecule compound combination used for preparing cartilage tissue engineering products or regenerative medicine cartilage seed cells and chondrocytes.
The methods of the invention are performed under conditions suitable for the production of induced chondrocytes, including, for example, the composition of the culture, concentration, temperature of culture, time of culture, and other conditions. Based on the full teaching of the prior art and in connection with the exemplary illustrations of the present invention, one skilled in the art can readily determine the above culture conditions without undue experimentation. The key is to select the desired inhibitory or activating cellular signaling pathways and to determine the order in which the cellular signaling pathways are to be affected. In addition, the concentration and other conditions of the small molecule compound or combination thereof can be adjusted adaptively based on the ranges provided by the present invention.
The mechanism of the invention is as follows: the invention activates the reprogramming endogenous transcription factor through the action of histone acetylation and methylation of differentiated cells, reduces the methylation level of cell genome DNA through inhibiting the activity of HMT, starts the expression of reprogramming related genes, and realizes the dedifferentiation of differentiated cells and the reprogramming of the differentiated cells into chondrocytes through the synergistic action of inhibiting a TGF-beta signal path, activating a WNT/beta-catenin signal path, activating a cAMP signal path, exciting an RA signal path, inhibiting the activity of PKC and the like.
There are a large number of reports in the art on small molecules suitable for different signaling pathways, and those skilled in the art are still constantly developing such molecules. In the present invention, the small molecule compound to be used is not particularly limited in structure or classification, but is required to have a function of inhibiting or activating lysine deacetylase, TGF- β, PKC, DNMT, HMT, JNK, ROCK, WNT/β -catenin, cAMP, RA (Retinoic acid). Therefore, the invention covers all molecules that can realize the corresponding inhibition or activation functions of lysine deacetylase, TGF-beta, PKC, DNMT, HMT, histone demethylase, JNK, ROCK, WNT/beta-catenin, cAMP, RA (Retinoic acid), and covers the alternatives that can realize the corresponding inhibition or activation functions of the above targets.
The invention has the following technical effects: according to the invention, the chondrocytes are prepared by inducing differentiated cells of human by stages through small molecular compounds, and each step can realize accurate quality control, so that standardized operation and large-scale production are facilitated. The method provided by the invention has wide donor source, and the patient can be used as the donor and can specifically obtain the chondrocyte required by basic research, clinical treatment or tissue engineering production in a short time. The method can be widely applied to basic medical research, clinical cell therapy and research and development of related human tissue engineering products, and has an industrial prospect.
Drawings
FIG. 1 is a schematic diagram of the method for transdifferentiation of human dermal fibroblasts into chondrocytes in example 1;
FIG. 2 is a cell morphology diagram of transdifferentiation of human dermal fibroblasts into chondrocytes induced by small molecule compounds, wherein A is a skin fibroblast; b is chondrocytes obtained by transdifferentiation according to the method of example 1, and stained with alcian blue; c is the chondrocyte obtained by transdifferentiation by the method in example 6, and staining with alcian blue was performed;
FIG. 3 is a graph showing the quantitative identification of molecular markers of chondrocytes from transdifferentiated chondrocytes, wherein HuFib represents untreated skin fibroblasts (negative control), HuiCH represents transdifferentiated chondrocytes, and HuCH represents chondrocytes obtained by in vitro induction of in vivo MSCs (positive control); after treatment with a combination of small molecular compounds, the resultant was differentiated for 14 days using a conventional cartilage inducing solution, and identified as a map A and a map B, which show highly expressed cartilage marker genes in hyaline cartilage, and a map C and a map D, which show highly expressed genes in hypertrophic cartilage. By identifying the expression amount, the chondrocytes prepared by using the small molecular compound combination rarely progress to the hypertrophic cartilage stage;
fig. 4 is a graph showing cell growth curves, and a graph a is an identification of collagen2a of transdifferentiated chondrocytes, which were treated with a combination of small-molecule compounds and then differentiated for 14 days using a conventional cartilage inducing solution. B is a graph showing the growth curve of skin fibroblasts isolated from adult human (chondrocytes prepared from the same as a raw material can be mass-produced in 10 consecutive passages under the condition of culture in a high-sugar culture medium containing 10% serum);
fig. 5 shows that human dermal fibroblasts (panel a, cell morphology before initial induction) induced the cell products (panel B, cell morphology after 17 days of induction) well after 17 days of the experimental method described in example 8, but the cell products (panel D, cell morphology after 17 days of induction) induced by mesenchymal stem cells isolated in vivo (panel C, cell morphology before initial induction) exhibited massive cell death. Thus, the chondrocytes produced by the method used in the invention are derived from differentiated cells (e.g., skin fibroblasts in example 8), and not from other types of stem cells (e.g., mesenchymal stem cells).
Detailed Description
The technical scheme of the present invention is further described in detail below with reference to the drawings and specific examples, but the present invention is not limited to the following experimental scheme.
Example 1
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 passage of cells, the number of passage of cells is between 6 th and 12 th passage, for inducing transdifferentiation into chondrocytes. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In an incubator.
2. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 12 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2. mu.M-20. mu.M) + Repsox (2-15. mu.M) + CHIR99021 (1. mu.M-10. mu.M) + VPA (0.5 mM-1.5 mM) + TTNPB (3. mu.M-8. mu.M) + AM580 (0.03-0.08. mu.M) + EPZ004777 (3-8. mu.M) + G06983 (1-15. mu.M) + Y-27632 (3-15. mu.M) + L-Ascorblin acid 2-phospate (0.15-0.25 mM), 10% fetal bovine serum (invitrogen) in the culture system can also be replaced by serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
3. Second stage of induction
Then changing to the third stage culture solution, acting for 3-10 days, at 37 deg.C and 5% CO2And culturing the cells under the environment. The third stage culture medium is cultured on the second stage culture medium by using a combination of a growth factor combination and a DNMT inhibitor or a combination of a growth factor and a DNMT inhibitor.
The growth factor combinations employed in this example include: BMP4(10-20ng/m L) + PDGF-AB (100-250ng/m L) + b-FGF (10-50ng/m L), wherein the DNMT inhibitor is specifically 5-Aza-2' -deoxycytidine (1u M-15 u M), and 10% of fetal calf serum in the culture system can be replaced by a serum substitute (invitrogen) at a concentration of 10-20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100nM dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
5. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
Example 2
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 cell passagesAnd (3) carrying out large-scale expansion, wherein the cell generation number is between 6 th generation and 12 th generation, and the cell generation number is used for carrying out transdifferentiation induction to the chondrocyte. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In an incubator.
2. Pretreatment of skin fibroblasts
2.1 when the transdifferentiation is started (Day0), completely replacing the basic culture solution into a first-stage culture solution, culturing the cells for 4-6 days, wherein the first-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 mug/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 muM-25 muM) + Repsox (2-15 uM) + CHIR99021(1 muM-10 muM) + VPA (0.5 mM-1.5 mM), wherein 10% fetal bovine serum can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used. At 37 ℃ 5% CO2And culturing the cells under the environment.
3. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 10 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2. mu.M-20. mu.M) + Repsox (2-15. mu.M) + CHIR99021 (1. mu.M-10. mu.M) + VPA (0.5 mM-1.5 mM) + TTNPB (3. mu.M-8. mu.M) + AM580 (0.03-0.08. mu.M) + EPZ004777 (3-8. mu.M) + G06983 (1-15. mu.M) + Y-27632 (3-15. mu.M) + L-Ascorblin acid 2-phospate (0.15-0. 0.25M M), 10% fetal bovine serum (invitrogen) can also be replaced by serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Second stage of induction
Then, the culture medium was changed to the third stage culture medium, the action time was 3 to 10 days, and the cells were cultured at 37 ℃ under 5% CO 2. The third stage culture medium is cultured in combination with a growth factor combination, a DNMT inhibitor or a combination of a growth factor and a DNMT inhibitor, on the basis of the second stage culture medium.
The growth factor combinations employed in this example include: BMP4(10-20ng/m L) + PDGF-AB (100-250ng/m L) + b-FGF (10-50ng/mL), wherein the DNMT inhibitor is specifically 5-Aza-2' -deoxycytidine (1u M-15 u M), and 10% of fetal bovine serum in the culture system can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
5. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100n M dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
6. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
Example 3
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 passage of cells, the number of passage of cells is between 6 th and 12 th passage, for inducing transdifferentiation into chondrocytes. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In the incubator。
2. Pretreatment of skin fibroblasts
2.1 when the transdifferentiation is started (Day0), completely replacing the basic culture solution into a first-stage culture solution, culturing the cells for 4-6 days, wherein the first-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 mug/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 muM-25 muM) + Repsox (2-15 uM) + CHIR99021(1 muM-10 muM) + VPA (0.5 mM-1.5 mM), wherein 10% fetal bovine serum can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used. At 37 ℃ 5% CO2And culturing the cells under the environment.
3. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 10 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 μ M-20 μ M) + Repsox (2-15 uM) + CHIR99021(1 μ M-10 μ M) + VPA (0.5 mM-1.5 mM) + TTNPB (3 μ M-8 μ M) + AM580 (0.03-0.08 μ M) + EPZ004777 (3-8 μ M) + G06983 (1-15 μ M) + Y-27632 (3-15 μ M) + L-Ascorblin acid 2-phospate (0.15-0. 0.25M M) + SP600125 (8-12 μ M), 10% fetal bovine serum in the culture system can also be replaced by serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Second stage of induction
Then, the culture medium was changed to the third stage culture medium, the action time was 3 to 10 days, and the cells were cultured at 37 ℃ under 5% CO 2. The third stage culture medium is cultured in combination with a growth factor combination, a DNMT inhibitor or a combination of a growth factor and a DNMT inhibitor, on the basis of the second stage culture medium.
The growth factor combination adopted by the embodiment comprises BMP4(10-20ng/m L) + PDGF-AB (100-250ng/m L) + b-FGF (10-50ng/m L), wherein the DNMT inhibitor is specifically 5-Aza-2' -deoxycytidine (1u M-15 u M), and 10% of fetal calf serum in the culture system can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
5. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100n M dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
6. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
Example 4
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 passage of cells, the number of passage of cells is between 6 th and 12 th passage, for inducing transdifferentiation into chondrocytes. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In an incubator.
2. Pretreatment of skin fibroblasts
2.1 when transdifferentiation is initiated (Day0), the basal medium is completely changed to the first stage medium, cells are cultured for 4-6 days, firstThe stage culture solution is: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 mug/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 muM-25 muM) + Repsox (2-15 uM) + CHIR99021(1 muM-10 muM) + VPA (0.5 mM-1.5 mM), wherein 10% fetal bovine serum can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used. At 37 ℃ 5% CO2And culturing the cells under the environment.
3. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 10 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 μ M-20 μ M) + Repsox (2-15 uM) + CHIR99021(1 μ M-10 μ M) + VPA (0.5 mM-1.5 mM) + TTNPB (3 μ M-8 μ M) + AM580 (0.03-0.08 μ M) + EPZ004777 (3-8 μ M) + G983 (1-15 μ M) + Y-27632 (3-15 μ M) + L-Ascorblin acid 2-phoshate (0.15-0. 0.25M M) + SP600125 (8-12 μ M) +5-Aza-2' -deoxymycin (1 μ M) + 10% fetal bovine serum (1-15 μ M) +10 μ M), and the fetal bovine serum (10-10% Vitrogen) can be replaced by the culture system; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Second stage of induction
Then changing to the third stage culture solution, acting for 3-10 days, at 37 deg.C and 5% CO2And culturing the cells under the environment. The third stage culture medium is cultured in combination with a growth factor combination, a DNMT inhibitor or a combination of a growth factor and a DNMT inhibitor, on the basis of the second stage culture medium.
The growth factor combination adopted by the embodiment comprises BMP4(10-20ng/m L) + PDGF-AB (100-250ng/m L) + b-FGF (10-50ng/m L), wherein the DNMT inhibitor is specifically 5-Aza-2' -deoxycytidine (1u M-15 u M), and 10% of fetal calf serum in the culture system can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
5. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100nM dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
6. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
Example 5
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 passage of cells, the number of passage of cells is between 6 th and 12 th passage, for inducing transdifferentiation into chondrocytes. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In an incubator.
2. Pretreatment of skin fibroblasts
2.1 when the transdifferentiation is started (Day0), completely replacing the basic culture solution into a first-stage culture solution, culturing the cells for 4-6 days, wherein the first-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ g/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 μ M-25 μ M) + Repsox (2-15 μ M) + CHIR99021 (1-10 μ M) + VPA (0.5-1.5 mM), 10% of fetal bovine serum in the culture system can also be replaced by serum replacement (invitrogen) with a concentration of 10-20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used. At 37 ℃ 5% CO2And culturing the cells under the environment.
3. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 10 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 μ M-20 μ M) + Repsox (2-15 uM) + CHIR99021(1 μ M-10 μ M) + VPA (0.5 mM-1.5 mM) + TTNPB (3 μ M-8 μ M) + AM580 (0.03-0.08 μ M) + EPZ004777 (3-8 μ M) + G983 (1-15 μ M) + Y-27632 (3-15 μ M) + L-Ascorblin acid 2-phoshate (0.15-0. 0.25M M) + SP600125 (8-12 μ M) +5-Aza-2' -deoxymycin (1 μ M) + 10% fetal bovine serum (1-15 μ M) +10 μ M), and the fetal bovine serum (10-10% Vitrogen) can be replaced by the culture system; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Second stage of induction
Then, the culture medium was changed to the third stage culture medium, the action time was 3 to 10 days, and the cells were cultured at 37 ℃ under 5% CO 2.
The third stage culture solution adopted in this embodiment comprises 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + forskolin (2. mu.M-20. mu.M) + Repsox (2-15. mu.M) + CHIR99021 (1. mu.M-10. mu.M) + VPA (0.5 mM-1.5 mM) + TTNPB (3. mu.M-8. mu.M) + AM580 (0.03-0.08. mu.M) + EPZ004777 (3-8. mu.M) + G06983 (1-15. mu.M) + Y-27632 (3-15. mu.M) + L-ascorbic acid 2-phospatite (0.15-0.25M M) +5-Aza-2' -deoxyytidine (1. mu.M-15. mu.M) + PDGF-10. mu.M) + FGF-3632 b (1-15. mu.M) + FGF-3632), the 10% fetal calf serum in the culture system can also be replaced by serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
5. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100n M dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
6. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
Example 6
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 passage of cells, the number of passage of cells is between 6 th and 12 th passage, for inducing transdifferentiation into chondrocytes. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In an incubator.
2. Pretreatment of skin fibroblasts
2.1 when the transdifferentiation is started (Day0), completely replacing the basic culture solution into a first-stage culture solution, culturing the cells for 4-6 days, wherein the first-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2. mu.M-25. mu.M) + Repsox (2-15. mu.M) + CHIR99021 (1. mu.M-10. mu.M) + VPA (0.5 mM-1.5 mM), 10% in the culture system%Fetal bovine serum can also be replaced by serum replacement (invitrogen) at a concentration of 10% to 20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used. At 37 ℃ 5% CO2And culturing the cells under the environment.
3. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 10 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 μ M-20 μ M) + Repsox (2-15 uM) + CHIR99021(1 μ M-10 μ M) + VPA (0.5 mM-1.5 mM) + TTNPB (3 μ M-8 μ M) + AM580 (0.03-0.08 μ M) + EPZ004777 (3-8 μ M) + G983 (1-15 μ M) + Y-27632 (3-15 μ M) + L-Ascorblin acid 2-phoshate (0.15-0. 0.25M M) + SP600125 (8-12 μ M) +5-Aza-2' -deoxymycin (1 μ M) + 10% fetal bovine serum (1-15 μ M) +10 μ M), and the fetal bovine serum (10-10% Vitrogen) can be replaced by the culture system; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Second stage of induction
Then, the culture medium was changed to the third stage culture medium, the action time was 3 to 10 days, and the cells were cultured at 37 ℃ under 5% CO 2.
The third stage culture solution used in this example includes 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + forskolin (2 μ M. about.20 μ M) + Repsox (2. about.15 uM) + CHIR99021(1 μ M. about.10 μ M) + VPA (0.5 mM. about.1.5 mM) + TTNPB (3 μ M. about.8 μ M) + AM580 (0.03. about.0.08 μ M) + EPZ004777 (3. about.8 μ M) + G06983 (1. about.15 μ M) + Y-27632 (3. about.15 μ M) + L-ascorbic acid 2-phospatite (0.15. about. 0.25M M) +5-Aza-2' -deoxyytidine (1 μ M. about.15 μ M) + bovine serum 10% replaced by 10% fetal bovine serum (10-10%) in a system; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
5. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100n M dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
6. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
Example 7
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 passage of cells, the number of passage of cells is between 6 th and 12 th passage, for inducing transdifferentiation into chondrocytes. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In an incubator.
2. Pretreatment of skin fibroblasts
2.1 when the transdifferentiation is started (Day0), completely replacing the basic culture solution into a first-stage culture solution, culturing the cells for 4-6 days, wherein the first-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 mug/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 muM-25 muM) + Repsox (2-15 uM) + CHIR99021(1 muM-10 muM) + VPA (0.5 mM-1.5 mM), wherein 10% fetal bovine serum can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.gml streptomycin (Sigma) may not be used. At 37 ℃ 5% CO2And culturing the cells under the environment.
3. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 10 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 μ M-20 μ M) + Repsox (2-15 uM) + CHIR99021(1 μ M-10 μ M) + VPA (0.5 mM-1.5 mM) + TTNPB (3 μ M-8 μ M) + AM580 (0.03-0.08 μ M) + EPZ004777 (3-8 μ M) + G06983 (1-15 μ M) + Y-27632 (3-15 μ M) + L-Ascorblin acid 2-phospate (0.15-0. 0.25M M) + SP600125 (8-12 μ M), 10% fetal bovine serum in the culture system can also be replaced by serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Second stage of induction
Then, the culture medium was changed to the third stage culture medium, the action time was 3 to 10 days, and the cells were cultured at 37 ℃ under 5% CO 2.
The third stage culture solution used in this example includes 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + forskolin (2 μ M. about.20 μ M) + Repsox (2. about.15 uM) + CHIR99021(1 μ M. about.10 μ M) + VPA (0.5 mM. about.1.5 mM) + TTNPB (3 μ M. about.8 μ M) + AM580 (0.03. about.0.08 μ M) + EPZ004777 (3. about.8 μ M) + G06983 (1. about.15 μ M) + Y-27632 (3. about.15 μ M) + L-ascorbic acid 2-phospatite (0.15. about. 0.25M M) +5-Aza-2' -deoxyytidine (1 μ M. about.15 μ M) + bovine serum 10% replaced by 10% fetal bovine serum (10-10%) in a system; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
5. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100n M dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
6. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
Example 8
1. Isolation of skin fibroblasts
1.1 obtaining a mass of skin tissue of about 1cm in diameter from a donor, isolating the skin fibroblasts by adherence, culturing the isolated cells in a basal medium which: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high sugar DMDM.
1.2 passage of cells, the number of passage of cells is between 6 th and 12 th passage, for inducing transdifferentiation into chondrocytes. The Day before the initiation of differentiation (Day-1), the cell density was 1-2.5X 104/cm2Culturing at 37 deg.C and 5% CO2In an incubator.
2. Pretreatment of skin fibroblasts
2.1 when the transdifferentiation is started (Day0), completely replacing the basic culture solution into a first-stage culture solution, culturing the cells for 4-6 days, wherein the first-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 mug/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 muM-25 muM) + Repsox (2-15 uM) + CHIR99021(1 muM-10 muM) + VPA (0.5 mM-1.5 mM), wherein 10% fetal bovine serum can be replaced by a serum substitute (invitrogen) at a concentration of 10% -20%; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used. At 37 ℃ 5% CO2And culturing the cells under the environment.
3. First phase induction of skin fibroblasts
After the treatment of the step 2, the culture solution is completely replaced by a culture solution of a second stage for cell culture, the culture time is 6 to 10 days, and the culture solution is cultured at 37 ℃ and 5 percent CO2And culturing the cells under the environment. The second-stage culture solution is as follows: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high-sugar DMDM medium (Gibco) + forskolin (2 μ M-20 μ M) + Repsox (2-15 uM) + CHIR99021(1 μ M-10 μ M) + VPA (0.5 mM-1.5 mM) + TTNPB (3 μ M-8 μ M) + AM580 (0.03-0.08 μ M) + EPZ004777 (3-8 μ M) + G983 (1-15 μ M) + Y-27632 (3-15 μ M) + L-Ascorblin acid 2-phospatate (0.15-0. 0.25M M) +5-Aza-2' -deoxytide (1 μ M-15 μ M), and 10% fetal bovine serum (10%) can be substituted for the 10% fetal bovine serum; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
4. Second stage of induction
Then, the culture medium was changed to the third stage culture medium, the action time was 3 to 10 days, and the cells were cultured at 37 ℃ under 5% CO 2.
The third stage culture solution used in this example includes 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) +100 μ G/ml streptomycin (Sigma) + high sugar DMDM medium (Gibco) + forskolin (2 μ M. about.20 μ M) + Repsox (2. about.15 uM) + CHIR99021(1 μ M. about.10 μ M) + VPA (0.5 mM. about.1.5 mM) + TTNPB (3 μ M. about.8 μ M) + AM580 (0.03. about.0.08 μ M) + EPZ004777 (3. about.8 μ M) + G06983 (1. about.15M) + Y-27632 (3. about.15 μ M) + L-ascorbic acid 2-phospahate (0.15. about. 0.25M M) +5-Aza-2' -deoxyytidine (1 μ M. about.15 μ M) + bovine serum 10% -10% replacement by 10% fetal bovine serum; 100U/ml penicillin (Sigma) and 100. mu.g/ml streptomycin (Sigma) may not be used.
5. Chondrogenic directed differentiation of cells
Then, the cells were cultured for 12 to 30 days in a conventional chondrogenic differentiation medium or a commercially available chondrogenic induction solution (seiko biotechnology limited) to obtain chondrocytes. Alternatively, after the first and second steps of induction of differentiated cells are performed, the cells are directly subjected to a chondrogenic differentiation medium of the fourth step or a commercially available chondrogenic induction liquid (seiko biotechnology limited). The chondrogenic differentiation solution is specifically: 10% fetal bovine serum (Hyclone) +100U/ml penicillin (Sigma) + 100. mu.g/ml streptomycin (Sigma) + high-sugar DMDM Medium (Gibco) +1 XTS +100n M dexamethone +10ng/ml TGF-beta3+0.17mM ascorbate-2-phosphate + 40. mu.g/ml proline
6. Detection of induced chondrocytes, detection of genes Col2a1, Aggrecan, sox9, Col10x, corresponding immunohistochemical staining, alcian blue staining.
The chondrocytes prepared in the above example and the relevant assays of chondrocytes are shown in fig. 1-5; the skin fibroblasts used in the examples may be replaced with other differentiated cells such as blood cells, fat cells, etc.
It is to be understood that within the scope of the present invention, the various features of the invention described above, as well as those specifically described below (e.g., in the examples), may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Claims (2)

1. A method for preparing chondrocytes by inducing fibroblasts is characterized in that the method comprises the steps of directionally inducing the fibroblasts for 6-12 days in a first stage, then directionally inducing the fibroblasts for 3-10 days in a second stage, replacing the fibroblasts with conventional chondrogenic differentiation culture solution or commercial osteogenic culture solution sold in the market, and culturing the fibroblasts for 12-30 days to prepare the chondrocytes;
the small molecules adopted in the first stage directional induction are selected from any one of the following items:
1）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate;
2）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+SP600125;
3）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+SP600125+5-Aza-2′-deoxycytidine;
4）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+ 5-Aza-2′-deoxycytidine;
the small molecules adopted by the second stage directional induction are selected from any one of the following items:
1）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+BMP4+PDGF-AB+b-FGF+5-Aza-2′-deoxycytidine;
2）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO06983+Y-27632+L-Ascorbin acid-2-phosphate+BMP4+PDGF-AB+b-FGF+5-Aza-2′-deoxycytidine+SP600125;
3）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate +5-Aza-2′-deoxycytidine。
2. a method for preparing chondrocytes by inducing fibroblasts is characterized by comprising the steps of pretreating before directional induction for 4-6 days, directionally inducing the fibroblasts for 6-10 days in a first stage, then directionally inducing the fibroblasts for 3-10 days in a second stage, finally replacing the fibroblasts with a conventional chondrogenic differentiation culture solution or a commercial osteogenic culture solution sold in the market, and culturing for 12-30 days to prepare the chondrocytes;
the small molecules adopted by the pretreatment are as follows:
Forskolin+Repsox+CHIR99021+VPA；
the small molecules adopted in the first stage directional induction are selected from any one of the following items:
1）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate;
2）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+SP600125;
3）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+SP600125+5-Aza-2′-deoxycytidine;
4）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+ 5-Aza-2′-deoxycytidine;
the small molecules adopted by the second stage directional induction are selected from any one of the following items:
1）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+BMP4+PDGF-AB+b-FGF+5-Aza-2′-deoxycytidine;
2）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate+BMP4+PDGF-AB+b-FGF+5-Aza-2′-deoxycytidine+SP600125;
3）、Forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+GO6983+Y-27632+L-Ascorbin acid-2-phosphate +5-Aza-2′-deoxycytidine。
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