CN112442481B

CN112442481B - Method for differentiating adipose-derived stem cells and umbilical cord blood stem cells in cooperation

Info

Publication number: CN112442481B
Application number: CN202011360830.7A
Authority: CN
Inventors: 刘欢; 李蒙
Original assignee: Henan Beike Biotechnology Co ltd
Current assignee: Henan Beike Biotechnology Co.,Ltd.
Priority date: 2020-11-27
Filing date: 2020-11-27
Publication date: 2021-09-17
Anticipated expiration: 2040-11-27
Also published as: CN112442481A

Abstract

The invention relates to a method for differentiating adipose-derived stem cells in cooperation with umbilical cord blood stem cells. The invention obtains the promoting active peptide which can specifically promote the FOXO3 high expression in the cord blood stem cells through screening and optimizing, and the polypeptide can promote the proliferation of the cord blood stem cells and the differentiation to the erythrocytes through improving the expression of FOXO 3; also provided is an improved method for increasing cord blood stem cell proliferation using adipose derived stem cells, the method comprising incubating adipose derived stem cells with cord blood stem cells. The expanded hematopoietic stem cells are further cultured in a specially prepared induction medium to obtain differentiated red blood cells. The invention can amplify hematopoietic stem cells at high speed and obtain red blood cells with better erythroid characteristics, can be used for subsequent clinical research on a large scale and has better application value.

Description

Method for differentiating adipose-derived stem cells and umbilical cord blood stem cells in cooperation

Technical Field

The invention relates to the field of biomedicine, in particular to a method for synergic differentiation of adipose-derived stem cells and umbilical cord blood stem cells.

Background

As a new donor for hematopoietic stem cell transplantation, the umbilical cord blood has the advantages of wide source, easy collection, no harm to the donor, low HLA matching requirement, high hematopoietic stem/progenitor cell (HSPC) content and the like. Moreover, cord blood is produced in a time period 4-5 weeks shorter than the time required to obtain hematopoietic stem cells from a matched unrelated donor. In addition, the disease recurrence rate after Umbilical Cord Blood Transplantation (UCBT) and the incidence of graft versus host disease are also low compared to hematopoietic stem cell transplantation from other sources. However, the biggest limitation of the clinical application of UCBT is the low total number of nucleated cells (TNC) extracted from cord blood. The research shows that: the fewer HLA-matched sites, the greater the amount of TNC required. Depending on the type of disease, the number of cord blood sufficient per serving is defined as: complete consensus of HLA sites>3×10⁷TNC/kg, 5 sites are met>4× 10⁷TNC/kg, 4 sites match>5×10⁷TNC/kg. The number of nucleated cells contained in cord blood is only 5% to 10% of the available concentration of nucleated cells in peripheral blood and bone marrow, which, if performed in low numbers, causes delayed recovery of neutrophils and increased risk of bacterial and viral infections. The double cord blood transplantation expands the range of umbilical cord blood transplantation for adults, but increases the incidence rate of graft-versus-host disease and prolongs the recovery time of plateletsAnd thus cause a series of problems.

In order to solve the problem of expansion of umbilical cord stem cells, currently, the umbilical cord blood is expanded by adding cytokines separately. Many studies have only demonstrated that different cytokines have a synergistic effect during HSC expansion. Although the number of nucleated cells and progenitor cells is increased and related molecules expressed on the cell surface can promote the homing effect of the cells, the cells are differentiated to different degrees through the amplification of the cell factors. Because HSCs rely on a local microenvironment in vivo and strict regulation of complex internal and external signals including transcription factors, cell cycle regulators, growth factors and adhesion molecules to maintain a balance between self-renewal and multipotential differentiation. Therefore, it is difficult to maintain the original dryness of HSC by in vitro expansion. Most research experiments also prove that the in vitro expanded cord blood HSPC has poor long-term implantation capability, and the proportion of the cord blood HSPC occupies the dominant position of implanted cells in a short term. From the current research, although the total amount of TNC is obviously increased by using the cytokine to expand the cord blood, the success rate of UCBT is not improved because the early progenitor cells are induced to be differentiated into cell groups with weak homing capacity while the total amount of TNC is expanded. Scientists therefore utilize specific small molecules to prevent differentiation of early progenitor cells during in vitro expansion of cord blood, and to make the expanded cells less differentiated, so as to have better bone marrow homing ability. Examples of the chelating agent include copper ion chelating agent copperchemical (TEPA, StemEx), NotchLigand, Nicotinamide (Nicotinamide), Aryl Hydrocarbon antioxidant (stemregen 1, SR1), and the like. Many trials of small molecule amplified HSPCs are underway in multicenter clinical trials, where StemEx has completed phase III trials.

The stem cell microenvironment is the microenvironment for stem cell growth, which plays an important role in maintaining HSC self-renewal and multipotential differentiation. Techniques to facilitate expansion of HSCs in vitro by mimicking the hematopoietic microenvironment are also becoming increasingly mature. MSC has important hematopoietic support for HSC mainly because: MSCs can provide a variety of in vitro expanded signaling pathways not found in HPC suspension cultures based on cytokine alone. MSCs can regulate HSCs by direct contact or secretion of endogenous cytokines, among other means. Moreover, expansion of HSPCs by MSC feeder cultures avoids the reduction in cell number caused by CD34 and CD133 sorting. Expansion of cord blood using bone marrow mesenchymal stem cells (BM-MSC) as feeder layers clinical trials in phase I/II have been completed by co-culturing cord blood with adherent BM-MSC, hematopoietic-related cytokines for 7d, followed by re-expansion of culture with cytokines alone for 7 d. Finally, the median increase of the number of CD34+ cells in the umbilical cord blood HSPC is 30 times of the median of the original cells.

However, at present, there is no study for improving the proliferation and erythrocyte differentiation of cord blood stem cells using adipose-derived stem cells.

Disclosure of Invention

The present invention provides an improved method for increasing the proliferation and erythrocyte differentiation of cord blood stem cells by using adipose derived stem cells.

The inventor previously screens and obtains two promoting active peptides capable of specifically promoting the high expression of FOXO3 in cord blood stem cells. The two polypeptides can promote the proliferation of cord blood stem cells and the differentiation of cord blood stem cells into erythrocytes by increasing the expression of FOXO 3.

In another aspect, there is provided an improved method for increasing cord blood stem cell proliferation using adipose stem cells, the method comprising incubating adipose stem cells with cord blood stem cells.

Further, the proliferation promoting step comprises taking one 24-well Transwell plate, carefully clamping the upper chamber containing the semipermeable membrane with sterile forceps, adding the adipose-derived stem cell suspension resuspended in the adipose-derived stem cell culture medium into the lower chamber, uniformly blowing, and carefully adding the cord blood stem cell suspension into the upper chamber with a pipette gun so that the upper chamber contains cells: the lower chamber contains cells in a ratio of 1:1 to 1: 10. Wherein umbilical cord hematopoietic stem cell culture medium: DMEM-F12 medium 90% + FBS 10% + SEQ ID NO: 1 polypeptide 50. mu.g/mL + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL. Adipose-derived stem cell culture medium: DMEM-F12 medium 44.5ml + Fetal Bovine Serum (FBS)5ml + penicillin-streptomycin 500ul and mixed well.

Further, the present invention provides an improved method for increasing the proliferation and differentiation of cord blood stem cells into erythrocytes using adipose-derived stem cells, comprising the steps of:

(1) and (3) proliferation steps: taking a 24-hole Transwell culture plate, carefully clamping an upper chamber containing a semipermeable membrane by using a sterile forceps, adding an adipose-derived stem cell suspension resuspended by an adipose-derived stem cell culture medium into a lower chamber of the upper chamber, blowing, uniformly mixing, and carefully adding an umbilical cord blood stem cell suspension into the upper chamber by using a pipette gun so that the upper chamber contains cells: the lower chamber contains cells in a ratio of 1:1 to 1: 10. Wherein umbilical cord hematopoietic stem cell culture medium: DMEM-F12 medium 90% + FBS 10% + SEQ ID NO: 1 polypeptide 50. mu.g/mL + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL. Adipose-derived stem cell culture medium: DMEM-F12 culture medium 44.5ml + Fetal Bovine Serum (FBS)5ml + penicillin-streptomycin 500ul mixing;

(2) and (3) differentiation steps: centrifuging the hematopoietic stem cells obtained by amplification in the step (1) to obtain cells, continuously carrying out red blood cell differentiation in an induction culture medium for 8 days, and then obtaining corresponding differentiated red blood cells; the induction medium comprises the following components: using StemBan SFEM as a basic culture medium, wherein the final addition concentrations are respectively SEQ ID NO: 50ng/mL of 1 polypeptide, 400ng/mL of SCF, 160ng/mL of IGF, 15IU/mL of Epo, 100 mu g/mL of transferrin, 10 mu g/mL of hyaluronic acid, 1mM of L-glutamine and 1.5 mu M of dexamethasone.

As the peptide of the present invention, there is provided an amino acid sequence peptide of SEQ ID NO. 1.

And providing a medium for promoting cord blood stem cell proliferation, the medium consisting of: DMEM-F12 medium 90% + FBS 10% + SEQ ID NO: 1 polypeptide 50. mu.g/mL + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL.

The peptide represented by the above SEQ ID NO: 1, which can be obtained by various methods widely known in the art. Detailed description: a method of producing the protein by gene recombination or a protein expression system, or a method of synthesizing the protein in vitro by chemical synthesis such as peptide synthesis, a cell-free protein synthesis method, and the like. More specifically, it can be synthesized by methods well known in the art such as automated peptide synthesizer, or can be produced by transgenic technology, but is not limited thereto. For example, a fusion gene encoding a fusion protein of a fusion partner and a peptide fragment of the present invention is prepared by transgenosis, and used to transform a host microorganism into a trait, and then expressed in the form of a fusion protein in the host microorganism, and then the peptide fragment of the present invention can be cleaved and isolated from the fusion protein by using a cleavage enzyme or a compound to produce a desired peptide fragment. For this purpose, the DNA sequence between the fusion partner and the peptide gene according to the invention, for example the protein cleavage enzymes CNBr such as Factor Xa or telomerase or hydroxytryptamine, encodes amino acid residues which are possibly cleaved by the compounds. Can be inserted.

With the above-described SEQ ID NO: 1, or an additional amino acid sequence designed for the specific purpose of increasing the residue half-life or peptide stability of a targeting sequence tag (tag) label.

Advantageous effects

The invention obtains two promoting active peptides capable of specifically promoting the high expression of FOXO3 in cord blood stem cells by screening and optimizing. The two polypeptides can promote the proliferation of cord blood stem cells and the differentiation to erythrocytes by increasing the expression of FOXO 3; also provided is an improved method for increasing cord blood stem cell proliferation using adipose derived stem cells, the method comprising incubating adipose derived stem cells with cord blood stem cells. The expanded hematopoietic stem cells are further cultured in a specially prepared induction medium to obtain differentiated red blood cells. The invention can amplify hematopoietic stem cells at high speed and obtain red blood cells with better erythroid characteristics, can be used for subsequent clinical research on a large scale and has better application value.

Drawings

FIG. 1: FOXO3 expression quantity result chart

FIG. 2: cell growth profile

FIG. 3: cytokine expression level results plot

FIG. 4: result graph of cell enucleation level

Detailed Description

The technical scheme of the invention is described by combining specific embodiments. The experimental materials not particularly emphasized in the following examples are all conventional experimental materials, and are not particularly required, and are all conventional materials readily available to those skilled in the art.

EXAMPLE 1 isolation of cord hematopoietic Stem cells

First, separating mononuclear cells from umbilical cord blood by using Ficoll lymphocyte separation liquid, adding 100 mu L of FcR blocking reagent and 100 mu L of CD34 immune microbeads into cell suspension, uniformly mixing, putting into a refrigerator, and incubating for 30min at 4 ℃. And washing with MACS solution to elute, and removing cell debris, mixed cells and the like. The column was removed and placed on a collection tube, MACS solution (LS column: 5ml) was added, a plunger was attached, and the adsorbed cells were washed out by rapid pushing down with force. The column was repeated once, after which the centrifugation force was 300g, the supernatant was removed by centrifugation for 10min, and the cells were resuspended in 500. mu.L of MACS solution. Cells were collected, cytometric, and CD34 cell purity was determined by flow cytometry. The human umbilical cord blood hematopoietic stem cells purified by the MidiMACS immunomagnetic bead method are detected by a flow cytometer, the percentage of CD34 cells is (93.2 +/-4.6)%, and the human umbilical cord blood hematopoietic stem cells accord with the characteristics of hematopoietic stem cells for later use.

Example 2 Effect of the Polypeptides in promoting expression of FOXO3

Converting SEQ ID NO: 1 was added to StemBan SFEM medium at a concentration of 0, 1ug/mL, 10ug/mL, 50ug/mL, 100ug/mL, respectively. The cells isolated in example 1 were aligned at 5X 10⁵cells/ml are inoculated in 1ml culture medium for suspension culture for 48h, and FOXO3 gene expression quantity detection is carried out. RNA of the same cell quantity is extracted, and simultaneously, the TaKaRa kit reversely transcribes the RNA into cDNA. RT-PCR detection was performed with the following primers. The FOXO3 primer sequence was: upstream 5'-CGGACAAACGGCTCACTCT-3', downstream 5'-GGACCCGCATGAATCGACTAT-3'; the GAPDH primer sequence is: upstream 5'-ACAACTTTGGTATCGTGGAAGG-3', downstream 5'-GCCATCACGCCACAGTTTC-3'. The obtained product is placed in a 7300HTRT-PCR instrument for amplification. The reaction conditions were set according to the TaKaRa kit conditions. Analysis was performed using the ABI7300 System SDSSSoft software, using 2^-ΔΔCtAnd (4) calculating by using the method. The experiments were repeated 3 times. The results are shown in FIG. 1.

After the polypeptide treatment is used, the gene expression level of FOXO3 is remarkably improved. Specifically, the concentration of the polypeptide is 100ug/mL, and the gene expression level is improved to (3.4 +/-0.3) times.

Example 3 isolation of adipose-derived Stem cells

Abdominal fat from healthy humans was cut into 0.1cm by 0.1cm size tissues and washed 3-5 times with PBS buffer. Digesting with collagenase I with the volume of 5 times of fat, repeatedly shearing fat tissue into paste, shaking at 37 ℃ for 30min, filtering the digested fat tissue into a centrifuge tube by using a 250ul nylon gauze, centrifuging at 1000r/min for 30min, removing supernatant and suspended fat fragments, suspending the precipitate in 00.075mmol/L potassium chloride, standing for 10min, centrifuging, removing injury, suspending the precipitate in DMEM culture solution containing 10% fetal calf serum, transferring the precipitate into a culture dish, placing the culture dish in a 37 ℃, 5% CO2 incubator, culturing at 100% humidity, replacing the culture solution every other day, after the cells are paved with 90% of the bottom area of the culture dish, digesting with 0.25% trypsin, and carrying out passage according to the proportion of 1: 3. Taking the third generation cell, and detecting the expression condition of the marker by using a flow cytometer. The results show that the markers CD90, CD105, CD44, CD73, CD146, CD166 and CD29 are all highly expressed, and the expression rate is higher than 96%; markers CD45, CD31, CD34 and HLA-DR are all under expressed; the experimental result shows that the adipose-derived stem cells are successfully separated.

Example 4 promoting Effect of adipose-derived Stem cells on cord blood Stem cells

(1) Co-culture experimental group: a24-well Transwell plate was removed, carefully removed from the upper chamber containing the semipermeable membrane with sterile forceps, and 600. mu.l of the adipose-derived stem cell suspension isolated in example 3, resuspended in adipose-derived stem cell culture medium, was added to the lower chamber and mixed by pipetting. 100ul of the cord blood stem cell suspension isolated in example 1 was carefully added to the upper chamber using a pipette gun. Such that the upper chamber of each well contains cells 10⁴The lower chamber contains 5X 10 cells⁴And (4) respectively. The total liquid amount per well was 700ul, and 3 multiple wells were provided. Wherein umbilical cord hematopoietic stem cell culture medium: DMEM-F12 medium 90% + FBS 10% + SEQ ID NO: 1 polypeptide 50. mu.g/mL + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL. Adipose-derived stem cell culture medium: DMEM-F12 medium 44.5ml + Fetal Bovine Serum (FBS)5ml + penicillin-streptomycin 500ul and mixed well.

(2) Co-culture peptide-free experimental group: getA24-well Transwell plate was carefully removed from the upper chamber containing the semipermeable membrane with sterile forceps, and 600. mu.l of the adipose-derived stem cell suspension isolated in example 3, which had been resuspended in adipose-derived stem cell culture medium, was added to the lower chamber and mixed by pipetting. 100ul of the cord blood stem cell suspension isolated in example 1 was carefully added to the upper chamber using a pipette gun. Such that the upper chamber of each well contains cells 10⁴The lower chamber contains 5X 10 cells⁴And (4) respectively. The total liquid amount per well was 700ul, and 3 multiple wells were provided. Wherein umbilical cord hematopoietic stem cell culture medium: DMEM-F12 medium 90% + FBS 10% + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL. Adipose-derived stem cell culture medium: DMEM-F12 medium 44.5ml + Fetal Bovine Serum (FBS)5ml + penicillin-streptomycin 500ul and mixed well.

(3) Umbilical cord hematopoietic stem single culture group: adding the umbilical cord hematopoietic stem cells separated in the example 1 into a 24-well plate, setting 3 multiple wells, adding 700ul umbilical cord hematopoietic stem cell culture medium into each well, and uniformly blowing and beating to ensure that each well contains 10 cells⁴Total amount of liquid 700 ul/well. Cord blood stem cell culture medium: DMEM-F12 medium 90% + FBS 10% + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL.

The proliferation of cord blood stem cells was measured by the CCK-8 method after culturing the above 3 groups for 1, 3, 5, and 7 days. Collecting cord blood stem cell samples of 3 groups, marking the cord blood stem cell samples in the collected EP tubes, placing the cord blood stem cell samples in a centrifuge, centrifuging at 1200rpm for 5min, discarding the supernatant, and resuspending to 100 ul/tube. The resuspended cord blood stem cell suspension in each EP tube was aspirated into 96-well plates, 100 ul/well, and the cell-derived groups per well were labeled. 100ul of cord blood stem cell culture medium without cells was plated in 96-well plates in 3 duplicate wells as controls. Under the condition of keeping out of the light, 10ul of CCK-8 reagent is added into each hole, the incubation is carried out for 5 hours in a carbon dioxide coating box at the temperature of 37 ℃, the hole plate cover is taken out and opened, and the OD value of each hole at the position of 450 nm is measured by using an enzyme labeling instrument. Each group had 3 replicates and the experiment was repeated 3 times. HSC proliferation curves were plotted. The results are shown in FIG. 3.

The growth curve shows that the number of all groups of umbilical cord blood stem cells increases along with the prolonging of the culture time, the umbilical cord blood stem cells enter a logarithmic growth phase from day 3, the umbilical cord blood stem cells are co-cultured with the adipose-derived stem cells to have a promoting effect on the proliferation of the umbilical cord stem cells, particularly the promoting effect is more obvious after the polypeptide is added, and the OD reaches 3.5 +/-0.3 on day 7, so that the umbilical cord stem cells have a better proliferation effect.

EXAMPLE 5 differentiation of expanded hematopoietic Stem cells into erythrocytes

And (3) centrifuging the hematopoietic stem cells obtained after the three groups of experiments in the example 4, harvesting the cells, continuing to perform red blood cell differentiation in an induction culture medium for 8 days, and then harvesting corresponding differentiated red blood cells for identification. The induction medium comprises the following components: using StemBan SFEM as a basic culture medium, wherein the final addition concentrations are respectively SEQ ID NO: 50ng/ml of 1 polypeptide, 400ng/ml of SCF, 160ng/ml of IGF, 15IU/ml of Epo, 100 mu g/ml of transferrin, 1mM of L-glutamine and 1.5 mu M of dexamethasone.

The cell is used for extracting RNA, carrying out reverse transcription to obtain cDNA, and detecting the expression conditions of a marker CD34 of the hematopoietic stem cell, erythroid development related transcription factors PU.1, Run X1, GATA1, SCL, globin zeta, globin epsilon and globin beta by RT-PCR (contrast). RT-PCR identification was performed using the primer set shown in FIG. 3. The PCR conditions were 95 ℃ 7min → (94 ℃ 50s → 58 ℃ 30s → 72 ℃ 1min) × 30 cycles → 72 ℃ 10 min. Specific primer sequences are shown below.

GATA-1 primer: f: CAGGTACTGCCCATCTCTAC, respectively; TCTGGCTACAAGAGGAGAAG is the ratio of R to R; SCL primer F: GCTGGCTTTTCTGTTTCCTG, respectively; TGACAACCCCAGGTCTTAGG is the ratio of R to R; PU.1 primer: CGACCATTACTGGGACTTCC is used as a reference material; TTCTTCTTCACCTTCTTGACC is the ratio of R to R; RUNX1 primer: ATGTGGTCCTATT TAAGCCAGCCC is used as a reference material; TCATCTGGCTGAAGACACCAGCTT is the ratio of R to R; CD34 primer: AAATXCCTTCCTCTGAGGCTGGA; AAGAGGCAGCTGGTGATAAGGGTT is the ratio of R to R; beta-globin primer: f: GGGCAGGTTGGTATCAAGGTTAC, respectively; r: GGGGAAAGAAAACATCAAGCG, respectively; epsilon-globin primer: f: AAGATGAATGTGGAAGAGGCTG, respectively; r: TTAGCAAAGGCGGGCTTGAG, respectively; ζ -globin primer: f: CCAAGACTGAGAGGACCATCATTG, respectively; r: AGGACAGAGGATACGACCGATAGG, respectively; beta-Actin: f: GATCCACATCTGCTGGAAGG, respectively; AAGTGTGACGTTGACATCCG is added. The results are shown in FIG. 4, based on the expression level of β -actin as the relative expression level.

The results showed that the expression of the hematopoietic stem cell gene CD34 gradually decreased with the increase of the culture time, indicating that the cells gradually differentiated toward maturation with the increase of the culture time. The expression of CD34 in the co-culture experimental group is reduced fastest, which shows that the polypeptide can promote the differentiation of the hematopoietic stem cells to the erythrocytes rapidly, particularly in the co-culture group, the expression level of CD34 is only 3 +/-0.5 which is much lower than that of the co-culture peptide-free experimental group, which shows that the polypeptide can promote the differentiation of the hematopoietic stem cells to the erythrocytes. In addition, the expression of the early hematopoietic transcription factor SCL in the experimental group of the application is continuously negative, which indicates that the cells in the induction system have already differentiated. In the experimental group, PU.1 is gradually reduced in expression along with the prolonging of the induction time, GATA-1 is continuously and highly expressed, and cells in the induction system are differentiated towards the erythroid. In addition, the expression of globin ζ in the embryonic period is gradually reduced, and the expression of globin β in the adult period is continuously increased, which shows that the erythrocyte in the culture system undergoes the transition from the globin expression in the embryonic period mode to the globin expression in the adult period mode, and the erythrocyte is gradually matured and differentiated. In particular, the expression level of the adult globin beta in the co-culture experimental group is improved by 1.42 times compared with that in the co-culture peptide-free experimental group.

Example 6 analysis of the enucleation of erythrocytes:

three groups of red blood cells obtained by differentiation in example 5 were analyzed by flow cytometry for the proportion of CD235a positive/Hoechst 33342 negative cells, which reflects the cell enucleation. The results are shown in FIG. 4.

As can be seen from the figure, the enucleation level of the erythrocytes obtained by differentiation of the three groups in example 5, particularly the cells in the co-culture experimental group, can reach 88 percent, and is obviously improved compared with the enucleation level in the prior art.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

Sequence listing

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Claims

1. An improved method for using adipose stem cells to enhance the proliferation and differentiation of cord blood stem cells into red blood cells, said method comprising the steps of:

(1) and (3) proliferation steps: taking a 24-hole Transwell culture plate, carefully clamping an upper chamber containing a semipermeable membrane by using a sterile forceps, adding an adipose-derived stem cell suspension resuspended by an adipose-derived stem cell culture medium into a lower chamber of the upper chamber, blowing, uniformly mixing, and carefully adding an umbilical cord blood stem cell suspension into the upper chamber by using a pipette gun so that the upper chamber contains cells: the lower chamber contains cells in a ratio of 1:1 to 1: 10; wherein umbilical cord hematopoietic stem cell culture medium: DMEM-F12 medium 90% + FBS 10% + SEQ ID NO: 1 polypeptide 50. mu.g/mL + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL; adipose-derived stem cell culture medium: DMEM-F12 culture medium 44.5ml + Fetal Bovine Serum (FBS)5ml + penicillin-streptomycin 500ul mixing;

(2) and (3) differentiation steps: centrifuging the hematopoietic stem cells obtained by amplification in the step (1) to obtain cells, continuously carrying out red blood cell differentiation in an induction culture medium for 8 days, and then obtaining corresponding differentiated red blood cells; the induction medium comprises the following components: using StemBan SFEM as basic culture medium, the following substances were added and brought to the following final concentrations in the culture medium: SEQ ID NO: 50ng/mL of 1 polypeptide, 400ng/mL of SCF, 160ng/mL of IGF, 15IU/mL of Epo, 100 mu g/mL of transferrin, 10 mu g/mL of hyaluronic acid, 1mM of L-glutamine and 1.5 mu M of dexamethasone.

2. An improved method for increasing the proliferation of cord blood stem cells by using adipose-derived stem cells is characterized in that the step of promoting the proliferation comprises the steps of taking a 24-hole Transwell culture plate, carefully clamping an upper chamber containing a semipermeable membrane by using sterile forceps, adding adipose-derived stem cell suspension which is resuspended by adipose-derived stem cell culture medium into a lower chamber of the upper chamber, blowing and uniformly mixing, and carefully adding cord blood stem cell suspension into the upper chamber by using a liquid transfer gun so that the upper chamber contains cells: the lower chamber contains cells in a ratio of 1:1 to 1: 10; wherein umbilical cord hematopoietic stem cell culture medium: DMEM-F12 medium 90% + FBS 10% + SEQ ID NO: 1 polypeptide 50. mu.g/mL + SCF15ng/mL + IL-330ng/mL + hyaluronic acid 10. mu.g/mL; adipose-derived stem cell culture medium: DMEM-F12 medium 44.5ml + Fetal Bovine Serum (FBS)5ml + penicillin-streptomycin 500ul and mixed well.

3. An accelerating active peptide capable of specifically accelerating high expression of FOXO3 in cord blood stem cells, the amino acid sequence of the accelerating active peptide is shown as SEQ ID NO: 1 is shown.