CN111848743A - Improved method for differentiation of epidermal stem cells into pancreatic cells - Google Patents
Improved method for differentiation of epidermal stem cells into pancreatic cells Download PDFInfo
- Publication number
- CN111848743A CN111848743A CN202010792164.8A CN202010792164A CN111848743A CN 111848743 A CN111848743 A CN 111848743A CN 202010792164 A CN202010792164 A CN 202010792164A CN 111848743 A CN111848743 A CN 111848743A
- Authority
- CN
- China
- Prior art keywords
- cells
- stem cells
- epidermal stem
- pancreatic
- epidermal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The invention provides an improved method for differentiating epidermal stem cells into pancreatic cells, which is characterized in that the differentiation of the cells into the pancreatic cells is realized by adding activity inducing peptide based on the epidermal stem cells, the differentiation time is short, the sources of the epidermal stem cells are wide, the cost is low, the preparation is simple and convenient, and the market application prospect is excellent.
Description
Technical Field
The present invention relates to the field of non-embryonic pluripotent stem cells, and in particular to the use of non-embryonic pluripotent stem cells to provide pancreatic cells and methods of producing and using them.
Background
An embryonic stem cell is a totipotent stem cell that under the appropriate conditions can differentiate into somatic cells of any tissue in the body. Lumelsky et al reported that embryonic stem cells could be induced to differentiate into islet beta cells by an in vitro five-step induction method, while Shi et al reported in their studies that embryonic stem cells could be induced into islet beta cells by a three-step method, and that these cells, when transplanted into diabetic mice, increased the weight, prolonged the survival time, and decreased blood glucose of the animals. However, since embryonic stem cells have a tumorigenic problem that has not been solved so far, there is a certain distance from clinical use.
As for pancreatic ductal-derived stem cells, Bonner-Weir et al reported in their research reports that CK 19-positive stem cells were present in the ductal gland of the pancreas, and that such cells could be amplified and differentiated into islet beta cells in large amounts in the presence of KGF and Matrigel. As for islet-derived stem cells, Zulewski et al reported that they isolated and cultured a cell expressing the neural stem cell marker nestin protein (nestin) from an islet mass of a normal pancreas, and this cell was greatly expanded in vitro and differentiated into pancreatic endocrine cells by the action of exendin-4, activinA, HGF, Betacellulin and nicotiamide.
Huang & Tang, 2003; the pancreatic stem cells disclosed in Zulewski et al, 2001 article are adult stem cells derived from pancreatic islets. The tissue used by Zulewski et al is normal human or murine pancreatic tissue. The application of this technique to the clinic still faces the problem of insufficient resources, since it is difficult to obtain a sufficient amount of normal human pancreatic tissue in practice.
In the prior art, there are other ways to obtain pancreatic cells. Methods for differentiating non-embryonic pluripotent stem cells into the pancreatic lineage are provided, for example, in CN 101310012A. The invention further provides non-embryonic pluripotent stem cells and progeny derived therefrom, thereby providing pancreatic cells to a subject. The method comprises contacting a cell expressing Ngn3 with one or both of nicotinamide or exendiM to produce a cell expressing insulin, the cell expressing Ngn3 having been prepared by: (1) contacting non-embryonic stem, non-germ, non-embryonic germ cells that express Oct-4 and telomerase and that can differentiate into ectodermal, endodermal and mesodermal cell types with a first agent that is activin a and a second agent that inhibits sonic hedgehog (SHH) activity to produce cells having increased expression of Pdx-I; and (2) contacting the cells with increased expression of Pdx-I with EGF and/or HGF to produce cells with increased expression of Ngn 3. However, the method is complex to prepare, and the success rate needs to be further improved.
CN1847394A discloses a method for inducing differentiation of embryonic stem cells into pancreatic cells, which comprises treating embryoid bodies formed from embryonic stem cells with Activin a and cis-retinoic acid in this order to differentiate them into pancreatic cells. However, the method uses the embryonic stem cells, and the embryonic stem cells are not suitable for large-scale popularization and application due to limited sources, so that the market application value is not high.
Therefore, the problem of insufficient pancreatic tissue sources in islet transplantation needs to be solved, a stem cell with a wide source needs to be found, and pancreatic cells can be obtained by adopting a simple induction mode to meet the social needs, so that the human is benefited.
Disclosure of Invention
The present invention provides an improved method for the differentiation of epidermal stem cells, which are more convenient to source, into established pancreatic cells in a shorter amount of time (7 days) and with a significantly increased yield (up to 97% yield) compared to prior art protocols.
Provided herein is a method of differentiating epidermal stem cells into pancreatic cells, the method comprising the steps of:
in a first step, epidermal stem cells were induced with activated stimulatory peptides in 1% Matrigel plated dishes as follows:
after two hours, the epidermal stem cells began to spread, at which time the medium was changed to DMEM medium containing 10% fetal bovine serum and 100ng/ml activin A (Sigma) for 24 hours, and then the epidermal stem cells were changed to DMEM medium containing 10% fetal bovine serum and 1-10mg/L of active stimulating peptides for 6-8 hours to differentiate the epidermal stem cells. Thereafter, the differentiated epidermal stem cells were then used with a serum containing 10% fetal bovine serum and 1X 10-6The induction was continued for 24 hours in mol/L DMEM medium for RA (Sigma) to obtain pancreatic precursor cells.
In the second step, islet precursor cells are expanded.
The differentiated epidermal stem cells were cultured in DMEM containing 10% fetal bovine serum and 10ng/ml bFGF (Sigma) for 3-5 days, and the expansion of pancreatic precursor cells derived from the epidermal stem cells was induced. During this time, most of the adherent cells assume an epithelial-like structure.
Third, Pancreatic beta cells (Pancreatic beta cells) are matured.
The cells were transferred to DMEM/F12 medium (Gibco-BRL) containing N2 and B27(Gibco-BRL) serum replacement (from Gibco-BRL, Inc., according to the instructions), 1ug/ml Lamin (Sigma), 10ng/ml bFGF and 10mmol/L nicotinamide (Sigma) and cultured for 3-5 days to promote the maturation of islet beta cells, thereby obtaining mature pancreatic beta cells.
The concentration of Activin A can be 50-300ng/ml culture medium, and the concentration of cis-retinoic acid is 1 × 10-7-1×10-5mol/L culture medium; the concentration of Activin A is preferably 100ng/ml of culture medium, and the concentration of cis-retinoic acid is preferably 1 × 10-6mol/L culture medium.
The Matrigel is 1% by mass.
The concentration of the bFGF is 8-12 ng/ml; preferably 10 ng/ml.
And culturing the pancreatic precursor cells obtained by amplification for 3-5 days by using a culture medium added with N2 and B27 serum substitute, laminin (laminin), basic fibroblast growth factor (bFGF) and nicotinamide (nicotinamide), so as to obtain mature islet beta cells.
In another aspect, the invention provides active stimulating peptides, which can specifically aim at the induction trend of insulin expression related genes in epidermal stem cells, can remarkably increase the differentiation speed and the differentiation capacity of the epidermal stem cells, and can enhance the differentiation capacity of the epidermal stem cells to pancreatic cells.
The amino acid sequences of the active stimulating peptides related to the invention are respectively shown in SEQ ID NO: 1-6.
Detailed Description
Example 1 preparation of epidermal stem cells
The skin of the foreskin after circumcision of healthy male (human) is taken to prepare epidermal cells and fibroblasts.
1. Separation of epidermal cells: washing skin 2cm long and 2mm wide with PBS containing antibiotics for 3 times; digesting in protease solution at 4 deg.c for 16 hr; taking out the skin, and stripping the epidermis and the dermis; collecting epidermal skin pieces, placing in 0.25% pancreatin/0.02% EDTA (1: 1) mixture, digesting at 37 deg.C for 15 min, and finishingStopping digestion, slightly blowing, filtering, collecting cell suspension, centrifuging, removing supernatant, adding fresh culture solution, resuspending cells, and adjusting cell concentration to 1 × 103/ml。
2. Preparation of trophoblast cells: collecting the dermis skin sheet after peeling epidermis and dermis layer, placing in 625U/ml collagenase solution, digesting, collecting fibroblast suspension, culturing fibroblast to spread about 80%, adding mitomycin C to final concentration of 10-6mol/L, incubation at 37 ℃ for 12 hours, taking out, digesting with 0.25% pancreatin at 37 ℃ for 5 minutes, terminating digestion, centrifuging for 5 minutes (1000 r/min), discarding the supernatant, and resuspending the cells for later use.
3. Preparation of extracellular matrix covering plates: culturing the epidermal cells in a dish until the cells are confluent for 13 days; after the epidermal cells are converged, the culture solution is aspirated and washed with sterile PBS; adding a mixed solution of ethylenediamine tetraacetic acid (10mmol/L), tris (hydroxymethyl) aminomethane hydrochloric acid (25mmol/L) and triton (1% (w/v)), and incubating at 37 ℃ (30 minutes) until cell lysis can be seen under a lens; washing with PBS; then 0.5mg/ml denatured bovine serum albumin was added and incubated at 37 ℃ for 1 hour to block non-specific adhesion; washing with PBS, adding serum-free culture solution, and incubating at 37 deg.C.
4. Screening and culturing of epidermal stem cells: and (3) adding 2ml of epidermal cell suspension into a prepared plate covered with extracellular matrix, culturing at 37 ℃ for 10 minutes, taking out, sucking liquid, washing with PBS until cell-free suspension is achieved, wherein the adherent cells are epidermal stem cells. Adding trophoblast cells (4 × 10)3/cm2) The cells were cultured in KSC medium, and the medium was changed every 2 days and every 3 days thereafter. Wherein the KSC culture medium contains 5% fetal calf serum and 1ng/ml basic fibroblast growth factor.
5. Subculturing: the epidermal stem cells were grown to confluence, and digested with a mixture of 0.25% pancreatin/0.02% EDTA (1: 1) at 37 ℃ for 6 minutes, to terminate the digestion; collecting cell suspension, centrifuging for 5 min, discarding supernatant, adding fresh culture medium, resuspending cells at 2 × 105The cells were inoculated in a flask containing feeder cells at a density of one ml, cultured in a 5% CO2 incubator at 37 ℃ and then subjected to immunohistochemical detection: cell swinging sheet100 percent of beta 1 integrin, 97 percent of keratin 19, 10 percent of keratin and negative of anti-vimentin, and the epidermal stem cell is determined to be obtained.
EXAMPLE 2 preparation of active stimulating peptides
Firstly, a random polypeptide library is constructed, wherein the random polypeptide is a random amino acid sequence containing less than thirty (including thirty) amino acids. The random polypeptide library of the invention is obtained by translation transformation of a corresponding DNA library (a DNA library for screening CPP). The random library of polypeptides of the invention is transformed by methods commonly used in the art. Such as chemical transformation and electroporation, see molecular cloning (second edition of chinese), written by j. And (2) carrying out mixed culture on the random polypeptide and the epidermal stem cells, wherein the basal medium is a DMEM medium containing 10% fetal bovine serum and 100ng/ml activin A (Sigma), and 42 stimulating peptides with strong capacity of stimulating the epidermal stem cells to differentiate into pancreatic cells are obtained according to the difference of differentiation index capacity of the epidermal stem cells, wherein the strong capacity is SEQ ID NO: 1-6, adopting a chemical synthesis method to synthesize the polypeptide, and carrying out subsequent experiments.
Example 3 differentiation of epidermal Stem cells into pancreatic cells
Epidermal stem cells prepared in example 1 were placed in a 1% Matrigel-plated culture dish and induced with active stimulating peptides as follows:
after two hours, the epidermal stem cells began to spread, at which time the medium was changed to DMEM medium containing 10% fetal bovine serum and 100ng/ml activin A (Sigma) for 24 hours, and then the epidermal stem cells were changed to DMEM medium containing 10% fetal bovine serum and 10mg/L active stimulatory peptides (6 different active peptides of SEQ ID NO: 1-6 were separately tested without adding the active peptides as a blank) for 6 hours to differentiate the epidermal stem cells. Thereafter, the differentiated epidermal stem cells were then used with a serum containing 10% fetal bovine serum and 1X 10-6The induction was continued for 24 hours in mol/L DMEM medium for RA (Sigma) to obtain pancreatic precursor cells.
The preliminarily differentiated epidermal stem cells obtained as described above were cultured in DMEM containing 10% fetal bovine serum and 10ng/ml bFGF (Sigma) for 5 days to induce the expansion of pancreatic precursor cells derived from the epidermal stem cells. During this time, most of the adherent cells assume an epithelial-like structure.
The adherent cells obtained in the previous step are transferred into a DMEM/F12 culture medium containing N2 serum substitute, B27 serum substitute, 1ug/ml Lamin (Sigma), 10ng/ml bFGF and 10mmol/L nicotinamine (Sigma) to be continuously cultured for 5 days, so that the islet beta cells are promoted to mature, and mature pancreatic beta cells are obtained.
Example 4 detection of differentiated mature pancreatic beta cells
First, detecting the expression of differentiated cell genes by RT-PCR
In order to detect the condition of the pancreatic endocrine cells, the expression condition of the marker genes of the pancreatic endocrine cells is detected by RT-PCR, and the specific method and the result are as follows:
total RNA was extracted from differentiated cells and control cells using RNA extraction kit. The cells were differentiated into mature pancreatic beta cells. Control cells were not supplemented with active stimulatory peptides and cells obtained in the third step without the addition of active stimulatory peptides and without induction with either of activin A and RA small molecules. The RNA was reverse transcribed to cDNA using MMLV reverse transcriptase. PCR was performed using ExTaq polymerase system. The PCR primers were as follows:
insulin 1: TAGTGACCAGCTATAATCAGAG and ACGCCAAGGTCTGAAGGTCC (288 bp);
hnf3 beta: ACCTGAGTCCGAGTCTGACC and GGCACCTTGAGAAAGCAGTC (345 bp);
pdx-1: CTTAGCGTGTCGCCACAGCCCTCCA and TCCAACAGCCGCCTTTCGTTATTCT (472 bp);
glut 2: GGATAAATTCGCCTGGATGA and TTCCTTTGGTTTCTGGAACT (299 bp);
isl 1: ATTTGCCACCTAGCCACAGCACC and CGCATTTGATCCCGTACAACCTG (335 bp);
β-actin：CCTGAACCCTAAGGCCAACCGTGAA
and ATACCCAAGAAGGAAGGCTGGAAAA (480 bp).
The results are shown in the following table: the expression level of beta-actin is used as the reference level.
TABLE 1 expression level of each gene in differentiated cells
As can be seen from the above table, the differentiation of epidermal stem cells into pancreatic cells can be well promoted by using the active stimulating peptides of the present invention together with activin A and RA, while the differentiation of embryonic stem cells into pancreatic cells can be promoted only by using activin A and RA, like the prior art, but the differentiation of epidermal stem cells into pancreatic cells cannot be promoted. Moreover, the gene expression level of the pancreatic cells obtained by differentiation is not much different from that of normal human pancreatic cells, and is basically the same, and it is presumed that the pancreatic cells can basically perform the same function.
Second, ELISA detection of insulin secretion
2X104The differentiated pancreatic cells were stimulated with PBS (20 mmol/L glucose) and PBS (5 mmol/L glucose) for 1 hour, and the insulin secretion was measured as follows:
from the above results, it can be seen that the insulin secretion of pancreatic cells is significantly higher than that of control group, and the difference in insulin secretion caused by different concentrations of glucose is significant, demonstrating that pancreatic islet-like cells respond to the change of glucose concentration. Meanwhile, as can be seen from the results, the pancreatic cells induced by the invention can produce slightly less insulin than the normal pancreatic cells at low glucose concentration, but can produce higher insulin secretion than the normal pancreatic cells at high glucose concentration, which indicates that the differentiated cells have better hyperglycemia stimulation responsiveness and production capacity.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Sequence listing
<110> Luoyang Xuan Zhi Biotech Co., Ltd
<120> improved method for differentiating epidermal stem cells into pancreatic cells
<160>6
<170>SIPOSequenceListing 1.0
<210>1
<211>26
<212>PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>1
Trp Ile Met Ile His Asp Ile Tyr His Trp Asn Glu Ile Ile Glu Asn
1 5 10 15
Asn Thr Ser Phe Met Asp Leu Pro Ser Arg
20 25
<210>2
<211>26
<212>PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>2
His Ile Pro Pro Trp Trp Cys Met Thr Thr Trp Pro Met Gly Gly Ala
1 5 10 15
Asp Asn Ser Cys Lys Gly Arg Gln Arg Met
20 25
<210>3
<211>26
<212>PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>3
Ser Asp His Phe Gly Arg Gln Glu Ser Asp Met Ala Asp Lys Pro Val
1 5 10 15
Thr Gly Thr Asn Ala Lys Ile Ser Asp Gln
20 25
<210>4
<211>26
<212>PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>4
Pro Pro Leu Ala Thr Ser Ser Phe Thr Glu Trp Phe Pro Met Leu Trp
1 5 10 15
Cys Gln Asn Trp Ser Gln Ser Glu Val Tyr
20 25
<210>5
<211>26
<212>PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>5
Ser Gln Asn Pro Gly Asp Pro Thr Trp Ile Pro Leu Ser Ala Pro Gln
1 5 10 15
Thr Cys Ser Ser His Trp Arg Met Val Ile
20 25
<210>6
<211>26
<212>PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400>6
Tyr Pro Asn His Trp Leu Gln Ser His Ser Gln Ala Leu Met Glu Thr
1 5 10 15
Arg Arg His Trp Thr Ile Glu His Ser Met
20 25
Claims (5)
1. An active stimulatory peptide for stimulating cell differentiation.
2. An active stimulating peptide having the sequence of SEQ ID NO: and 6.
3. Use of the active stimulating peptides of any of claims 1-2 for promoting differentiation of epidermal stem cells into pancreatic cells.
4. A method for differentiating epidermal stem cells into pancreatic cells specifically comprises the following steps:
in a first step, epidermal stem cells were induced with activated stimulatory peptides in 1% Matrigel plated dishes as follows:
two hours later, the spreading of the epidermal stem cells was started, at which time the culture medium was changed to DMEM medium containing 10% fetal bovine serum and 100ng/ml of actin A for 24 hours, and then the epidermal stem cells were changed to DMEM medium containing 10% fetal bovine serum and 1-10mg/L of the active stimulatory peptides as claimed in any one of claims 1-2 to differentiate the epidermal stem cells, after which the differentiated epidermal stem cells were then cultured with DMEM medium containing 10% fetal bovine serum and 1 x10-6Continuing to induce the cells for 24 hours in a DMEM culture medium of mol/L RA to obtain pancreatic precursor cells;
secondly, expanding islet precursor cells;
culturing the differentiated epidermal stem cells in DMEM containing 10% fetal bovine serum and 8-12ng/ml bFGF for 3-5 days, inducing the expansion of pancreatic precursor cells derived from the epidermal stem cells, during which most adherent cells assume an epithelial-like structure;
third, Pancreatic beta cells (Pancreatic beta cells) are matured.
5. The method of claim 4, wherein the bFGF is provided at a concentration of 10 ng/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010792164.8A CN111848743B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811023578.3A CN109053866B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010792164.8A CN111848743B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811023578.3A Division CN109053866B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111848743A true CN111848743A (en) | 2020-10-30 |
| CN111848743B CN111848743B (en) | 2021-05-11 |
Family
ID=64759286
Family Applications (6)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811023578.3A Active CN109053866B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010792171.8A Active CN111848745B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010808780.8A Active CN111875674B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010792164.8A Active CN111848743B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010792168.6A Active CN111848744B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010808793.5A Active CN111875675B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
Family Applications Before (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811023578.3A Active CN109053866B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010792171.8A Active CN111848745B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010808780.8A Active CN111875674B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
Family Applications After (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010792168.6A Active CN111848744B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
| CN202010808793.5A Active CN111875675B (en) | 2018-09-03 | 2018-09-03 | Improved method for differentiation of epidermal stem cells into pancreatic cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (6) | CN109053866B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113462636B (en) * | 2021-08-05 | 2023-11-14 | 合肥滴碧云生物科技有限公司 | Improved method for differentiating epidermal stem cells into liver cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1847394A (en) * | 2005-04-15 | 2006-10-18 | 北京大学 | Method of inducing embryo stem cell to differentiate toward pancreatic cell |
| CN101580818A (en) * | 2009-03-09 | 2009-11-18 | 暨南大学 | Method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof |
| CN101603027A (en) * | 2008-06-12 | 2009-12-16 | 西北农林科技大学 | External evoked human marrow mesenchymal stem cell is divided into the method for insulin-like cell |
| CN105087468A (en) * | 2015-09-01 | 2015-11-25 | 东南大学 | Method for effectively inducing stem cells to differentiate into pancreas islet beta cells |
Family Cites Families (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1253558C (en) * | 2003-09-02 | 2006-04-26 | 中国人民解放军第四军医大学口腔医学院 | Separation and culturing method of human epidermis stem cell |
| CN1637137A (en) * | 2004-01-06 | 2005-07-13 | 中国人民解放军军事医学科学院野战输血研究所 | Method of inducing stem cell to differentiate to insulin-like cell and its application |
| CN1772885B (en) * | 2004-11-11 | 2010-12-01 | 中国人民解放军军事医学科学院野战输血研究所 | Method of inducing stem cell to differentiate into pancreatic island-like cell and application of pancreatic island-like cell |
| CN101173249A (en) * | 2007-10-17 | 2008-05-07 | 暨南大学 | Method for inducing human fetal liver mesenchyma stem cell to pancreatic islet beta sample cell differentiation |
| KR101025880B1 (en) * | 2008-10-23 | 2011-03-30 | 공희숙 | Method for the differentiation of human adult stem cells into insulin-secreting cells |
| CN102311940A (en) * | 2010-07-09 | 2012-01-11 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing differentiation of stem cells into islet-like cells |
| CN102433300A (en) * | 2011-11-25 | 2012-05-02 | 厚朴生物科技（苏州）有限公司 | Method for constructing pancreatic stem cell line from human insulin and differentiating to insulin secretion cell |
| CN102517248A (en) * | 2011-12-30 | 2012-06-27 | 中日友好医院 | In-vitro induced pancreas-islet-like structure forming method |
| CN102618491B (en) * | 2012-03-21 | 2014-04-09 | 协和干细胞基因工程有限公司 | Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution |
| CN102618500A (en) * | 2012-03-21 | 2012-08-01 | 天津科技大学 | Method for inducing human mesenchymal stem cells to differentiate into insulin-secreting cells in vitro |
| CN108004199B (en) * | 2012-04-30 | 2022-03-18 | 大学健康网络 | Methods and compositions for generating pancreatic progenitor cells and functional beta cells from hPSCs |
| CN108103006A (en) * | 2012-06-08 | 2018-06-01 | 詹森生物科技公司 | Differentiation of the human embryo stem cell to pancreatic endocrine cell |
| CN103146754A (en) * | 2013-02-04 | 2013-06-12 | 南通大学附属医院 | Method for differentiation of polygenic-modification induced pluripotent stem (iPS) cells into insulin secretion cells |
| CN103194424A (en) * | 2013-03-28 | 2013-07-10 | 于涛 | Method for inducing embryonic stem cell into pancreatic tissue-like cells |
| CN103740641A (en) * | 2013-12-26 | 2014-04-23 | 广州爱菲科生物科技有限公司 | Inducing culture medium and inducing method thereof for inducing human adipose-derived mesenchymal stem cell to be differentiated into islet-like cell |
| CN103756954A (en) * | 2014-01-22 | 2014-04-30 | 青岛中天干细胞工程有限公司 | Inducer for inducing human adipose derived stromal cells into insulin-secretion cells, induction medium and method |
| CN105462913A (en) * | 2014-09-05 | 2016-04-06 | 深圳市北科生物科技有限公司 | Method for inducing human umbilical cord mesenchymal stem cells to be differentiated into pancreatic beta cells |
| CN104263697B (en) * | 2014-09-18 | 2017-08-04 | 胡振波 | A kind of method that inducing culture and induction human adipose mesenchymal stem cells generate insulin secretory cell |
| CN104726395B (en) * | 2015-03-20 | 2018-03-23 | 深圳市人民医院 | It is a kind of to induce the method that people's induced multi-potent stem cell directed differentiation is pancreatic cell |
| CN104862268A (en) * | 2015-05-27 | 2015-08-26 | 贵州北科泛特尔生物科技有限公司 | Culture medium for separating and inducing human adipose stem cells into pancreatic beta cells and use method of culture medium |
| CN104988110A (en) * | 2015-07-08 | 2015-10-21 | 深圳爱生再生医学科技有限公司 | Method for transforming umbilical cord mesenchymal stem cells into islet cells |
| CN105062952A (en) * | 2015-08-06 | 2015-11-18 | 深圳爱生再生医学科技有限公司 | Method for transformation of mesenchymal stem cells into islet cells |
| CN105670986A (en) * | 2015-11-23 | 2016-06-15 | 王意忠 | Culture medium for inducing human umbilical cord mesenchymal stem cells to differentiate into islet-like cells and induction method therefor |
| CN106676056A (en) * | 2016-11-08 | 2017-05-17 | 里程 | Method for inducing differentiation from umbilical cord mesenchymal stem cells to insulin secretion cells |
| CN106854638B (en) * | 2017-01-19 | 2019-12-27 | 天晴干细胞股份有限公司 | Method for inducing mesenchymal stem cells to differentiate into islet-like cells |
| CN107267447A (en) * | 2017-06-18 | 2017-10-20 | 广东博溪生物科技有限公司 | A kind of enrichment method of epidermal stem cells |
| CN107460158A (en) * | 2017-08-10 | 2017-12-12 | 河南省银丰生物工程技术有限公司 | A kind of inducing umbilical cord mesenchymal stem is divided into the method to form insulin |
-
2018
- 2018-09-03 CN CN201811023578.3A patent/CN109053866B/en active Active
- 2018-09-03 CN CN202010792171.8A patent/CN111848745B/en active Active
- 2018-09-03 CN CN202010808780.8A patent/CN111875674B/en active Active
- 2018-09-03 CN CN202010792164.8A patent/CN111848743B/en active Active
- 2018-09-03 CN CN202010792168.6A patent/CN111848744B/en active Active
- 2018-09-03 CN CN202010808793.5A patent/CN111875675B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1847394A (en) * | 2005-04-15 | 2006-10-18 | 北京大学 | Method of inducing embryo stem cell to differentiate toward pancreatic cell |
| CN101603027A (en) * | 2008-06-12 | 2009-12-16 | 西北农林科技大学 | External evoked human marrow mesenchymal stem cell is divided into the method for insulin-like cell |
| CN101580818A (en) * | 2009-03-09 | 2009-11-18 | 暨南大学 | Method for inducing the differentiation of mesenchymal stem cells of human embryo livers into islet beta-like cells and stably expressing insulin and special induced liquid thereof |
| CN105087468A (en) * | 2015-09-01 | 2015-11-25 | 东南大学 | Method for effectively inducing stem cells to differentiate into pancreas islet beta cells |
Non-Patent Citations (2)
| Title |
|---|
| CHARLES E MURRY等: "Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development", 《CELL》 * |
| KEVIN A D"AMOUR等: "Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells", 《NAT BIOTECHNOL》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109053866B (en) | 2020-12-15 |
| CN111848744B (en) | 2021-07-23 |
| CN111875675A (en) | 2020-11-03 |
| CN111848743B (en) | 2021-05-11 |
| CN111875675B (en) | 2021-10-01 |
| CN109053866A (en) | 2018-12-21 |
| CN111848745A (en) | 2020-10-30 |
| CN111875674A (en) | 2020-11-03 |
| CN111848744A (en) | 2020-10-30 |
| CN111848745B (en) | 2022-01-18 |
| CN111875674B (en) | 2021-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101978044B (en) | Single pluripotent stem cell culture | |
| CN101952415B (en) | The differentiation of human embryo stem cell | |
| ES2665434T3 (en) | Differentiation of pluripotent stem cells using human feeder cells | |
| CN102695790A (en) | Pluripotent stem cells | |
| IL181767A (en) | Scalable process for cultivating undifferentiated stem cells in suspension | |
| EP2502988A1 (en) | A novel population of multipotent cardiac precursor cells derived from human blastocysts derived stem cells | |
| US20210054358A1 (en) | Method for making pluripotent stem cells | |
| KR101918817B1 (en) | Method for differentiation of hepatocyte derived from human stem cells and Hepatocyte | |
| CN101724602B (en) | Method for the differentiation of human adult stem cells into insulin-secreting cells | |
| Fang et al. | Differentiation of embryoid‐body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro 1 | |
| Meng et al. | The differentiation and isolation of mouse embryonic stem cells toward hepatocytes using galactose-carrying substrata | |
| H Parsons et al. | Patents on technologies of human tissue and organ regeneration from pluripotent human embryonic stem cells | |
| Zhou et al. | Genetic modification of primate amniotic fluid-derived stem cells produces pancreatic progenitor cells in vitro | |
| CN109053866B (en) | Improved method for differentiation of epidermal stem cells into pancreatic cells | |
| CN115975914A (en) | Method for inducing pluripotent stem cells by reprogramming of chemical small molecule drugs | |
| RU2430158C1 (en) | Method for differentiation of adult human stem cells in insulin-secreting cells | |
| KR101766372B1 (en) | Method for Inducing Ectodermal Differentiation of Embryoid Bodies Derived from Human Pluripotent Stem Cells by CXCR2 Stimulation | |
| Lee et al. | Clump-passaging-based efficient 3D culture of human pluripotent stem cells under chemically defined conditions | |
| KR20090050022A (en) | A method for differentiation of pluripotent stem cells to definitive endoderm cells | |
| JP6956984B1 (en) | Blastocyst preparation method | |
| Zhu et al. | Differentiation of Human umbilical cord-derived mesenchymal stem cells to melanocyte, Osteogenic and Neural cells in vitro | |
| CN101517069A (en) | A novel population of multipotent cardiac precursor cells derived from human blastocysts derived stem cells | |
| WO2015013875A1 (en) | Method for culturing totipotential stem cells | |
| AU2010201431A1 (en) | Method for the differentiation of human adult stem cells into insulin-secreting cells | |
| CA2701014A1 (en) | Method for the differentiation of human adult stem cells into insulin-secreting cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210422 Address after: No. 2069, fufu Road, Jingyue Development Zone, Changchun City, Jilin Province Applicant after: Jilin Huirong Biotechnology Co.,Ltd. Address before: 471000 Ludu Yuefu Building 1 Unit 1-1404, South of Qianjiang Road, West of Chang Xiamen Street, Luoyang Economic and Technological Development Zone, Henan Province Applicant before: LUOYANG XUANZHI BIOLOGICAL TECHNOLOGY Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder |
Address after: Floors 1-2, Office Building 1, Changchun Zhongbang Technology Development Co., Ltd., No. 999 Qingtianshu Street, Jingyue Development Zone, Changchun City, Jilin Province, 130117 Patentee after: Jilin Huirong Biotechnology Co.,Ltd. Address before: No. 2069 Fufu Road, Jingyue Development Zone, Changchun City, Jilin Province, 130122 Patentee before: Jilin Huirong Biotechnology Co.,Ltd. |
|
| CP02 | Change in the address of a patent holder |