WO2006034602A1

WO2006034602A1 - Methods for porliferation of hematopoietic stem/progenitor cell in vitro

Info

Publication number: WO2006034602A1
Application number: PCT/CN2004/001109
Authority: WO
Inventors: Xuetao Pei; Wei Shi; Dongmei Wang; Yanhua Li; Yunfang Wang; Fang Yan
Original assignee: Beijing Institute Of Transfusion Medicine
Priority date: 2004-09-28
Filing date: 2004-09-28
Publication date: 2006-04-06
Also published as: CN100572528C; CN101018855A

Abstract

The present invention relates to methods for proliferation of hematopoietic stem/progenitor cells in vitro. Feeder cells with Wnt3a gene introduced into can be used as cultured layers in the methods provided by the invention. Wnt3a gene may be introduced into and expressed in feeder cells, and it is proved in this study that the feeder layers secrete the active Wnt3a protein in natural form. Compared to directly adding purified Wnt3a protein, the methods of the present invention allow more stable effect of proliferation, more convenient application and less cost. A large scale of proliferation in vitro of hematopoietic stem cells may be performed, which may benefit the solution to the problem interfering the clinic hematopoietic stem cell transplant, i.e. the lack of cell donors.

Description

Method for external expansion of hematopoietic stem/progenitor cells

The present invention relates to a method of expanding hematopoietic stem/progenitor cells in vivo.

Background technique

Clinical hematopoietic stem cell transplantation has become a treatment for malignant, non-malignant hematologic malignancies and bone marrow remodeling in high-dose cancer chemotherapy patients. To obtain the amount of cells needed for treatment, many scientists have attempted to expand hematopoietic stem/progenitor cells by ex vivo, including the use of word cells and the combination of various cells. However, the methods used at present have limited the number of hematopoietic stem cells expanded, and the ability of hematopoietic cells to self-renew in the process of in vitro expansion is diminished and differentiated. This still cannot solve the serious shortage of donors from hematopoietic stem cell transplantation. The problem.

Hematopoietic stem/progenitor cells have the characteristics of self-renewal and rebuilding blood. Reya confirmed that the Li T signal pathway plays a crucial role in the process of hematopoietic stem/progenitor cell self-renewal. The protein encoded by the Wnt gene is a class of secreted glycoproteins consisting of 350 to 400 amino acids, including 23 or 24 conserved cysteines and approximately 50% at the carboxy terminus of the protein. Increasing its expression level not only can amplify the number of hematopoietic stem/progenitor cells (>100 times), but also reduce its differentiation; it does not affect its ability to rebuild blood. Willert found that the specificity of the Wnt3a protein to amplify stem/progenitor cells is inextricably linked to its own lipid-modifying structure. The purified Wnt3a protein is very prone to palmitoylation under external conditions, so that the purified protein loses its activity and thus cannot be used to amplify dry and progenitor cells. Nature. 2003 May 22 ; 423 (6938) : 448-52. Epub 2003 Apr 27) .

At present, in the study of activating hematopoietic stem/progenitor cells, bone marrow stromal cells (primary mouse stromal cells and stromal cell lines enrolled in ATCC, MS-5, TC-1, etc.) are used as trophoblasts through cell contact. The action and its secreted cytokines cooperate with other cytokines to produce an amplification effect on hematopoietic/progenitor cells.

Invention disclosure

The method for in vitro expansion of hematopoietic stem/progenitor cells provided by the present invention is to culture hematopoietic stem/progenitor cells by using a feeder cell into which a Wnt3a gene is introduced as a trophoblast.

The Wnt3a gene is a nucleotide sequence encoding a protein having the amino acid residue sequence of SEQ ID NO: 2 in the Sequence Listing. Sequence 2 in the sequence listing consists of 352 amino acid residues.

The Wnt3a gene may have one of the following nucleotide sequences:

1) a DNA sequence of SEQ ID No _: 1 in the Sequence Listing;

2) a DNA sequence having 90% or more homology with the DNA sequence defined by SEQ ID No _: L in the sequence listing, and encoding the amino acid residue sequence of SEQ ID NO _: 2 in the sequence listing;

3) A nucleotide sequence which hybridizes under high stringency conditions to the DNA sequence defined by SEQ ID NO: 1 in the Sequence Listing.

Wherein, the high stringency condition is that after hybridization, the membrane is washed at 65 ° C with a solution containing 0.1 X SSPE (or 0.1 X SSC), 0.1% SDS.

SEQ ID No _: 1 in the Sequence Listing consists of 1059 bases, and its coding sequence is from bases 1 to 1059 at the 5' end.

The feeder cells can be bone marrow stromal cells.

The bone marrow stromal cells may be derived from humans and may also be derived from all other vertebrate animals. Includes primary bone marrow stromal cells and established myeloid stromal cells, such as MS-5, TC-l o

The Wnt3a gene can be introduced into a feeder cell by a liposome or a viral vector.

The viral vector includes a vector capable of transfecting an animal cell such as an adenovirus vector, a retrovirus vector, and an adeno-associated virus.

In the above method, the Wnt3a gene can be introduced into a feeder cell by the recombinant adenovirus vector pAd-Wnt3a.

In the above method, the Wnt3a gene can be introduced into a word cell by the recombinant retroviral vector pMSCV-Wnt3a.

In order to obtain better results, the introduced gene flTnt3a 18Gy irradiated feeder cells were ^6Q Co Y rays as feeder layer.

In order to obtain a stable amplification effect, the feeder cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

DRAWINGS

Figure 1 shows the PCR amplification of Wnt3a

Figure 2 is an electropherogram of the recombinant virus plasmid pAd-Wrrt3a

Figure 3 is an electrophoresis map of Pac I for pAd-Wnt3a recombinant virus plasmid digestion. Figure 4 is a photo of GFP expression in transfected 293 cells. Figure 5 shows the expression profile of Wnt3a protein by Western-blot assay. Figure 6 is the electrophoresis map of restriction endonuclease digestion of pMSCV-Wnt3a recombinant retroviral vector by Hpa I and BamH I.

Figure 7 shows the results of virus titer determination of pMSCV-Wnt3a-infected NIH3T3 cells. Figure 8 shows the expression of Wnt3a mRNA in PT67 cells and NIH3T3 cells by RT-PCR. Figure 9 shows the expression profile of Wnt3a in RT-PCR.

Figure 10 shows the purity curve of CD34+ cells detected by flow cytometry.

Figure 11 shows the CFC colony formation assay of Wnt3a-expressing bone marrow stromal cells as a trophoblast. Figure 12 shows the HPP-CFC colony formation of Wnt3a-expressing bone marrow stromal cells as a wave-culture layer.

The best way to implement the invention

Example 1. Establishing a hematopoietic stem/fine trophoblast

I. Construction of recombinant adenoviral vector

1. Recombinant adenoviral plasmid produced by homologous recombination in bacteria

(1) Acquisition of Wnt3a gene

Using mouse placental cDNA as a template, primers were synthesized based on the sequence of Wnt3a-cDNA coding region and introduced with Xbal, Hpa I and BamH I restriction sites. The primer sequences and their polymerase chain reaction (PCR) conditions were as follows: J

5, -TCT AGA GTT AAC^TGGCTCCTCTCGGATACCT-3

P2 5' -|AGA TCT GGA TCC|CTACTTGCAGGTGrGCACGT- 3,

The 20 μΐ reaction system consists of: lul cDNA (lOOng/ul), PI and P2 each 0.5 μm (20 μΜ), 1. 5 μΐ dNTP (2.5 mM), 0. 25 μ1 La Taq DNA polymerase (TakaRa, 5 ι/μ1) , 2μ1 5 X GC Buffer I (TakaRa), 14. 75μ1 sterilized water. The reaction conditions were: denaturation at 94 ° C for 3 min; 94 ° C for 45 s, 56 ° C for 45 s, 72 ° C for 2 min, 28 cycles; 72 ° C for 7 min. The electrophoresis results are shown in Fig. 1. In Fig. 1, the channel 1 is the target gene, the lane M is the DNA marker (DL2000), and the arrow indicates the Wnt3a electrophoresis band of the target gene, which is 1059 bp in size. The amplified fragment was cloned and cloned into pGEM-T easy vector (Promega, USA) to obtain the cloning plasmid T-Wnt3a, and the gene sequence was prayed. The result showed that the amplified Wnt3a gene has SEQ ID NO: 1 in the sequence listing ( Nucleotide sequence of 1059 bases, the coding sequence of which is from bases 1 to 1059 of the 5' end, and the amino acids of sequence 2 in the sequence listing Residue sequence.

The T-Wnt3a plasmid was digested with restriction endonuclease Xbal, and the Wnt3a gene fragment was inserted into pAdTrack (pAdTrack from AdEasy Vector System (Quantum)

At the Xbal site of the Biotechnologies vector, the recombinant plasmid pAdTrack-Wnt3a ₀ was obtained and sequenced and analyzed. The open reading frame was correct and no frameshift was observed. The cloned target gene sequence was correct, and the recombinant plasmid pAdTrack-Wnt3a was successfully constructed.

After taking Lug _P AdTrack-Wnt3a linearized with Pme I, a 1% Qiongyue sugar gel electrophoresis was carried out, and a target fragment of 100 bp was recovered by gel, dephosphorylated with calf intestinal alkaline phosphatase, and ethanol precipitated for use. According to the method provided in the AdEasy Vector System instructions, the recombinant plasmid pAdEasy-1 was transferred into BJ5183 strain, and BJ51 83 strain containing pAdEasy-1 was picked to prepare highly competent competent bacteria, and then lug linearized pAdTrack-Wnt3a The bacterium was transferred to the BJ5183 strain containing pAdEasy-1, and cultured in a LB plate of 50 mg/L kanamycin at 37 ° C for 16-20 h, picking the clone, extracting the plasmid, and performing a 0.7% agarose gel electrophoresis. The result is shown in Fig. 2, indicating that the size of the plasmid was 43,000 bp, and a recombinant virus plasmid was obtained, which was named pAd-Wnt3a. Transfer pAd_Wnt3a to DH5 a. In Fig. 2, lane 1 is recombinant plasmid 1, lane 2 is recombinant plasmid 2 (recombinant plasmid 1, recombinant plasmid 2 is different from pAd-Wnt3a, respectively), and lane M is DNA marker (DL15000), and the arrow indicates Plasmid electrophoresis band.

2. Identification of the target gene in the recombinant virus plasmid pAd- Wnt3a

The recombinant plasmid pAd-Wnt3a was digested with Pac I, and the results were shown in Figure 3. It was shown that two bands of 39000 bp and 4500b were obtained, and the recombination was successful according to the characteristics of adenovirus. In Fig. 3, lane 1 is the result of pAd-Wnt3a electrophoresis by Pac I enzyme, and lane M is DNA marker (DL15000). The arrow indicates the electrophoresis band of pAd-Wnt3a recombinant virus plasmid which was digested with Pac I.

3. Packaging of recombinant virulence vector in 293 cells, purification of virus and titer determination Plasmid DNA extracted from DH5 alpha bacterium containing recombinant viral plasmid pAd-Wnt3a in step 1.

10 ug, linearized with Pac I. 293 cells were transfected with lipofectAMINE (GIBC0) according to the manufacturer's instructions. After 7-10 days, observation with a fluorescence microscope revealed that GFP expression was observed in most 293 cells (Fig. 4). Then, the cells were harvested, and the cells were lysed by freezing and thawing four times at -70 ° C / 37 ° C, and the virus-containing supernatant was centrifuged to re-infect 293 cells to amplify the recombinant virus, and after 3 days, the cells were collected and repeatedly frozen and thawed three times. . Using CsCl density concentration ultracentrifugation method The virus was purified and the concentrated virus stock was diluted in different proportions. Add 400 μ ΐ dilution to the 293 cell culture flask, incubate for 3 hours at 37 ° C, change the fresh medium for 18-24 h, count the number of GFP positive cells under a fluorescence microscope, and calculate the disease according to the following formula. Titer: Virus titer = 6?? Cell positive number X virus supernatant dilution factor / 0. 4 ml (pfu / ml). The result showed a virus titer of 3.16 X 10 ⁷ pfu/L.

Second, the establishment of stromal cell trophoblast

1. Isolation and culture of primary mouse stromal cells

Two male BABL/c mice, 4 weeks old, were sacrificed by cervical dislocation and 70% ethanol was sterilized for 20-30 minutes. The femur and tibia were taken to remove the soft tissue and osteophyte attached to the bone. The separated femur and 15 bones were transferred to another sterile dish, and the bone marrow was punched out with a syringe of 5 ml of PBS, and subjected to a 200-mesh filter for 3 minutes, and centrifuged at 1500 rpm for 4 minutes. The precipitated cells were resuspended in a 10 ml glass centrifuge tube, and 2×10 ⁶ cells were seeded in a plastic flask. The fluid was changed for the first time 7 days after inoculation. After that, 3-4 days of fluid change one owe, 7-10 days passage. After 3 - 4 generations, spare.

2. Establish efficient and stable expression of Wnt3a-derived bone marrow stromal cells

Discard the culture medium in the flask and add 1- 2 ml of a-MEM medium. Then, lOOul the packaged venom into the cell, place it in the carbon dioxide incubator for 3 hours, add 2IL1 fresh medium, incubate for 72 hours, and observe the green fluorescent protein table under the fluorescence microscope.

Bnt3a-transfected bone marrow stromal cells were expanded in vitro. 5. OX IOVml was inoculated into 24-well plates. After the cells were more than 90% confluent, they were exposed to ⁶ °Co γ-rays for 18 Gy. After 48 hours of culture, the cells were lysed to anti-Wnt3a. From the R&D company, a horseradish peroxidase-labeled rabbit anti-mouse IgG (purchased from Zhongshan Co., Ltd.) was used as a secondary antibody for Western-blot experiments. As shown in Figure 5, Wnt3a protein was detected. expression. It was demonstrated that bone marrow stromal cells transfected with Wnt3a were obtained, thereby establishing trophoblast cells. Lane 1 in Figure 5 is a bone marrow stromal cell that has not been transfected with Wnt3a, and lane 2 is a bone marrow stromal cell transfected with Wnt3a.

Example 2. Establish a trophoblast that amplifies hematopoietic stem/progenitor

Construction and identification of recombinant retroviral plasmid pMSCV-Wnt3a

1. Construction of recombinant retroviral plasmid pMSCV-Wnt3a

The T-Wnt3a plasmid of Example 1 was extracted with restriction endonucleases Hpa I and BamH I, and the desired fragment was inserted into the Hpa I and Bgl II sites of the pMSCVneo vector (purchased from Clontech) to obtain the gene of interest. Retroviral vector PMSCV-Wnt3a, recombinant plasmid After double digestion with Hpa I and BamH I, the bands of 5.15 and lkb were obtained by TAE agarose gel electrophoresis, which were consistent with the expected results. The positive and reverse sequencing and analysis showed that the open reading frame was correct. , no frame shift changes have occurred. The recombinant gene of pMSCV-Wnt3a was constructed successfully (Fig. 6). In Figure 6, lane 1 is the electrophoresis result of pMSCV-Wnt3a double digestion with Hpa I and BamH I. Lane M is DNA marker. (DL2000), the arrow refers to the Wnt3a band of the target gene, which is 1059 bp in size.

2. Establishment and screening of retroviral-producing cell lines

PT67 cells (purchased from Clontech) were cultured to 95% confluence in DMEM/F12 medium (complete medium) containing 10% fetal calf serum. 5 μg of pMSCV-Wnt3a was added to 250 μl of serum-free medium, and LipofectamineTM 2000 (purchased from Invitrogen) was added to 10 μl of 250 μl of the above culture solution, and the mixture was incubated at room temperature for 20 min. The PT67 cells were washed twice with the culture solution, and the DNA-LipofectamineTM 2000 mixture diluted with 1 ml of the culture solution was slowly dropped. After 6 hours of culture, lml of the culture medium containing 20% fetal bovine serum was added, and the culture was replaced after 24 hours of continuous culture. After transfection for 48 h, cells were passaged at 1:15 and added to 600 g/ml G418 for 14 days until colony formation of resistant cells. Large healthy colonies were selected, expanded with complete culture medium, supernatant was collected, filtered through a filter of 0.45 um, and frozen for storage.

3. Determination of recombinant retrovirus pMSCV-Wnt3a virus titer and establishment of PT-Wnt3a cell line

Good growth state 24h NIH3T3 cells were cultured to 60% confluence, corresponding to the viral solution prepared in ^10-2, ^10-4, for 10 - ^6-fold diluted, was added 1, 5-dimethyl - 1, 5 H dinitrogen "--Methylene polybromide (polybrene) to a final concentration of 8 g / ml. Pour the cell culture medium, separately absorb 1 ml of diluted virus solution into the culture dish, routinely culture for 5h, add 2ml of fetal bovine serum The culture medium was routinely cultured for 24 hours, and the fresh medium containing 500μ _§ /ιη1 G418 was replaced, and the medium was changed once every 3 days. After 14 days of culture, the episodes were observed to be visible to the naked eye. The culture solution was decanted, fixed with pure formaldehyde, and stained with Jimuza. Automatic counting by cloning counter, multiplied by dilution factor to obtain virus titer

(cfu). A total of ⁶ packaging cells with a virus titer of > 10 ⁶ cfu were selected, and the packaging cells with the highest titer (8.8×10 ⁷ cfu/ml) were screened (Fig. 7), and high titers were expressed to express Wnt3a after expansion. The packaging cell line PT-Wnt3a. In Figure 7, A: 10 - ² times dilution; B: 10 - 咅 dilution;

C: 10 - ⁶ fold dilution.

4. Detection of wild-type virus in recombinant retrovirus liquid NIH3T3 polyclonal obtained by virus titer in the above steps was expanded for 48 h with complete culture solution. After centrifugation, the supernatant was again infected with NIH3T3 cells, and 500 ug/ml G418 was added for screening. After 5 days, all the cells died. It is suggested that PT67 cells do not produce a detectable helper virus, and retroviruses prepared therefrom can infect humans and other mammalian cells and can be widely used and safe.

5. Expression and identification of recombinant retrovirus

The synthetic primers were designed based on the sequence of the Wnt3a-cDNA coding region and subjected to reverse transcription PCR (RT-PCR) reaction. Primer sequence P3: 5, - ATGGCTCCTCTCGGATACCT- 3', P4: 5' - CTACTTGCAGGTGTGCACGT-3'. Total RNA from PT67 cells and NIH3T3 cells that had not been transfected with pMSCV-Wnt3a was extracted by TRIzol reagent for RT-PCR and agarose gel electrophoresis. A 1059 bp amplified band was observed, while control cells not transfected with pMSCV-Wnt3a were observed.

(NIH3T3 cells) showed no positive bands, confirming that Wnt3a gene is transcribed in PT-Wnt3a cell line and NIH3T3 cells infected with pMSCV-Wnt3a (Fig. 8). In Figure 8, lane 1 is human placental lysate (positive control), lane 2 is PT-Wnt3a cells, lane 3 is PT67 cells, lane 4 is NIH3T3 cells infected with pMSCV-Wnt3a, and lane 5 is NIH3T3 cells.

Second, the establishment of trophoblast

For 20 h before viral infection, vaccination IX 10 ⁵ separate cultured primary murine bone marrow stromal cells in 6-well plates, grown to 40% confluence routine, respectively, through a nylon filter of 0. 45um reversal rate was collected by filtration recombinant virus The supernatant was infected with mouse bone marrow stromal cells, and _P MSCV-Wnt3a was repeatedly infected 3 times (24 h each time). The fresh medium containing 700 ug/ml G418 was replaced and screened every 3 days. After 14 days of culture, the single resistant clones were expanded and cultured. The total RNA of the resistant clonal cells was extracted with TRIzol reagent, and P3 and P4 were used as primers according to the conventional method. The results of RT-PCR and agarose gel electrophoresis showed that the desired band of 1059 bp was obtained as shown in Fig. 9, thereby demonstrating that a stable expression strain of Wnt3a has been established and can be used as a trophoblast cell. In Figure 9, lane 1 is the RT-PCR amplification result of the resistant clone, and lane M is the DNA marker (DL2000).

Example 3. Wnt3a-expressing bone marrow stromal cells transfected with recombinant adenoviral vector as a trophoblast for expansion of hematopoietic stem/progenitor

First, the acquisition of hematopoietic stem/progenitor cells

(1) Separation of cord blood mononuclear cells (MNC)

Materials and reagents

Umbilical cord blood is taken from the umbilical cord of a healthy full-term pregnancy. PBS: each dissolved in deionized water 1L 8. 0g NaCl, 0. 2g KC1, 1. 44g Na 2 HP0 4, 0. 24g KH 2 P0 4, adjusted with NaOH or HC1 pH7 4..

0. 5, methyl cellulose: lg methyl cellulose was added to 200mL of normal saline, after autoclaving, put it into a 4 Ό refrigerator to dissolve it, and shake it well before use.

1. The fresh umbilical cord blood of heparin anticoagulation is mixed with PBS in a volume ratio of 1:1, and then mixed with 0.5% methylcellulose in a volume ratio of 4:1, and allowed to stand at room temperature for 30 min, until the red blood cells are naturally When the sedimentation is clear to the limit, the supernatant is aspirated and placed in a 50 mL centrifuge tube, and centrifuged at room temperature for 1 min at 10,000 rpm for 5 min;

2. Discard the supernatant, add lOmLPBS to suspend the cells, and wash the cells as above;

3. Discard the supernatant and resuspend the cells in 5 mL PBS;

4. Add 5 mL of human leukocyte separation solution pre-equilibrated to room temperature in a 10 mL centrifuge tube.

[Ficoll- Hypaque solution, (1· 077 ±0. 0002) g/L], then slowly add 5 mL of the cell suspension along the tube wall and centrifuge at room temperature for 1 min at 1500 rpm. It can be seen that the top to bottom is divided into PBS, MNCs, Ficoll- Hypaque solution, and four layers of red blood cells;

5. Collect the interface C cell layer, centrifuge at room temperature, l, 000 rpm for 5 min, discard the supernatant; 6. Use lmL CD ₃₄ + cell separation buffer (PBS + 0.5% BSA + 2 mmol / L EDTA, pH 7.2) The cells were suspended, transferred to a 1.5 mL Ep tube, and counted.

(ii) Isolation of CD ₃₄ ⁺ cells from Li C (using the miniMACS separation system)

1. Every 10 ⁸ cord blood mononuclear cells (Li C) were suspended in 300 w L 4 ° C preset CD ₃₄ ⁺ cell separation buffer, and 100 μL non-specific blocking antibody FcR blocking agent was added and mixed. Then, IOO L magnetic beads-conjugated CD ₃₄ monoclonal antibody (FcR blocking agent, magnetic bead-conjugated CD ₃₄ monoclonal antibody were purchased from Miltenyi Biotec), mixed, and incubated at 6-12 ° C for 30 min;

2. Add 500 μl CD ₃₄ + cell separation buffer, centrifuge at 4 ° C, 2, OOO rpm for 3 min, discard the supernatant;

3. Resuspend the cells with 1 mL of degassed CD ₃₄ + cell isolation buffer to prepare a single cell suspension; 4. Fix the MACS separation column to the MACS magnetic field (MACS separation column and magnetic field are purchased from

In Miltenyi Biotec, rinse the separation column with degassed 2 mL CD ₃₄ + cell separation buffer;

5. Slowly attach the single cell suspension to the separation column to avoid air bubbles. After it naturally flows out, wash the unbound cells with 500 PL degassing CD ₃ cell isolation buffer for 4 times;

6. The separation column was removed from the magnetic field and eluted with IraL CD ₃₄ + cell separation buffer under pressure to collect CD ₃₄ + cells. count. (3) Purity analysis

The immunomagnetic beads-labeled cells were purified by magnetic field twice and divided into two groups. One group was the test group, and the purity of the isolated CD34+ cells was detected by PE-labeled anti-CD34 antibody (purchased from Miltenyi Biotec), and the other group was In the control group, PE-labeled anti-mouse IgG (purchased from Zhongshan) was incubated with the isolated CD34+ cells. The purity of CD34+ cells was 97.74% by flow cytometry (Fig. 10).

The surface of the isolated hematopoietic stem cells expresses CD34.

Where the test group is

Therefore, the purity of the isolated CD34' cells was measured by PE-labeled anti-CD34 antibody (purchased from Mi ltenyi Biotec), and the control group was incubated with PE-labeled anti-mouse IgG (purchased from Zhongshan) and the isolated CD34' cells. .

Second, the expansion of hematopoietic stem / progenitor cells

The hematopoietic stem/progenitor cells were expanded and expanded using the trophoblast established in Example 1 as follows:

1. Co-cultivation

CD ₃₄ + cells were added to contain 50 ng/ml recombinant human stem cell factor (rhSCF, purchased from peprotech), 10 ng/ml rhlL-3 (recombinant human interleukin-3, purchased from peprotech) and 20 ng/ml rhFL (recombinant human) Flt3 ligand, purchased from peprotech, Inc., early hematopoietic cell long-term medium MyelocultTM H5110 (purchased from Stem cel l), seeded in 24-well plates, cell density 1. 0 X 107 ml, 1 ml culture system per well. The trophoblast cells in the well plates of the test group were the trophoblast cells established in Example 1. The control group was bone marrow stromal cells that did not transfect Wnt3a. Three holes per group. The 24-well plates were placed in a 37 ° C, 5 % C0 ₂ incubator for 3 weeks, and the cells were changed half a week for 3 weeks.

2, mixed colony culture

From the wells that were continuously cultured for 3 weeks in step 1, 2000-8000 CD ₃₄ + cells were inoculated with 0.5 mL of 5 ug/L GM-CSF (granulocyte-macrophage colony-stimulating factor, purchased from peprotech), 5 ug/ L IL-3, 50 ug/L SCF (stem cell growth factor, purchased from peprotech), 2 U/mL EP0 (erythropoietin, purchased from peprotech), 5 X 10 ⁵ mol/L 2-ME (β-mercaptoethanol) , purchased from peprotech), 10% FBS, 10% HS (horse serum, purchased from peprotech) in methylcellulose premixed medium MethoCultTM SF (purchased from Stem Cell), per 24 well plates Holes 0. 5mL were added, cultured to 16 days to count Various colonies. The cell count was performed using a hemocytometer plate, and the average value was counted three times. Colony counts were directly observed under an inverted microscope (Nikon), and more than 50 cells were counted as one colony, and the average of 3 parallel wells was taken. The colony density is the number of colonies formed per 10 ⁴ cells. The results shown in Fig. 11 show that the number of colonies formed by the CD ₃₄ + cells cultured in the trophoblast cells established in Example 1 as the trophoblast (experimental group) was increased by 1.8% compared with the control group. 3. 2 times, in Figure 11, the left picture is the control, and the right picture is the experiment group.

3. Colony culture and counting of high proliferative potential colony forming cells (HPP-CFC)

5毫升纤维素纤维素预混混合中。 In the wells of the continuous culture for 3 weeks, 2000-8000 CD ₃₄ + cells were inoculated into 0. 5mL methylcellulose premixed medium

5中中(31611^611 Company, H4434), added in 0.5 ml per well of a 24-well plate, and cultured to 14 d, add 0.2 mL of IMDM containing 20% cord blood plasma, continue to culture for 28 days, The cell colonies with a diameter > 0.5 leg (cell number > 50,000) under an inverted microscope (Nikon) were taken as one HPP-CFC, and the average of three parallel wells was taken. The HPP-CFC content, ie the number of HPP-CFC colonies formed per 10 ⁴ cells. The results shown in Figure 12, to show one embodiment of trophoblast cells established as feeder Example (experimental) culture of CD ₃₄ + HPP- CFC colonies set cell density of 2. 2-4. 3 times the control group. Each of the figures in Fig. 12 is a colony of different shapes observed under a microscope.

Industrial application

The present invention uses a stem cell which introduces the Wnt3a gene and expresses Wnt3a as a trophoblast, and it has been experimentally confirmed that the trophoblast secretes an active native Wnt3a protein. Compared with the Wnt3a protein directly added and purified, not only the amplification effect is stable, but also the application is convenient, and the cost is low, and the hematopoietic stem cells can be massively expanded in vitro, thereby facilitating the problem of serious shortage of donors in clinical hematopoietic stem cell transplantation. The establishment of this method is expected to provide sufficient ideal target cells for clinical bone marrow transplantation, treatment of hematological diseases, cell therapy and gene therapy of hematopoietic stem/progenitor cells, and provide assistance for studying hematopoietic regulation and exploring stem cell self-renewal mechanisms. It also provides new theories and methods for the amplification and clinical application of other stem cells; at the same time, the establishment of this method will have an excellent application prospect in regenerative medicine based on genetic engineering, cell engineering and even tissue engineering, and It will produce huge social and economic benefits.

Claims

Claim

I. A method for expanding hematopoietic stem/progenitor cells in vitro by using a stem cell into which a Wnt3a gene is introduced as a trophoblast to culture hematopoietic stem/progenitor cells.

The method according to claim 1, wherein the Wnt3a gene is a nucleotide sequence encoding a protein having an amino acid residue sequence of SEQ ID NO: 2 in the sequence listing.

3. A method according to claim 2, wherein: said 13& gene can have one of the following nucleotide sequences:

1) a DNA sequence of SEQ ID No _: 1 in the Sequence Listing;

2) a DNA sequence having 90% or more homology with the DNA sequence defined by SEQ ID No _: 1 in the sequence listing, and encoding the amino acid residue sequence of SEQ ID NO _: 2 in the sequence listing;

4. The method according to claim 1, wherein: said feeder cells are bone marrow stromal cells.

5. The method of claim 4, wherein: said bone marrow stromal cells comprise primary bone marrow stromal cells and established bone marrow stromal cells.

The method according to any one of claims 1 to 5, wherein the Wnt3a gene is introduced into a feeder cell by a liposome or a viral vector.

7. The method according to claim 6, wherein: the viral vector comprises an adenoviral vector, a retroviral vector, and an adeno-associated virus.

8. The method according to claim 7, wherein: in the method, the Wnt3a gene is introduced into a feeder cell by recombinant adenoviral vector pAd-Wnt3a.

9. The method according to claim 7, wherein: in the method, the Wnt3a gene is introduced into a feeder cell by recombinant retroviral vector pMSCV-Wnt3a.

The method according to any one of claims 1 to 5, wherein in the method, the word cell into which the Wnt3a gene is introduced is irradiated with ^6D Co Y-rays as a trophoblast.

II. The method according to claim 6, wherein in the method, the feeder cell into which the Wnt3a gene is introduced is irradiated with ^6Q Co Y-rays at 18 Gy as a trophoblast.

The method according to claim 7, wherein in the method, the feeder cell into which the Wnt3a gene is introduced is irradiated with ⁶ Gy γ ray at 18 Gy as a trophoblast.

13. The method according to claim 8, wherein in the method, the feeder cell into which the Wnt3a gene is introduced is irradiated with ⁶ Gy γ ray at 18 Gy as a trophoblast.

The method according to claim 9, wherein in the method, the feeder cell into which the Wnt3a gene is introduced is irradiated with ⁶ Gy y ray at 18 Gy as a trophoblast.

The method according to any one of claims 1 to 5, wherein the feeder cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

The method according to claim 6, wherein the feeder cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

17. The method according to claim 7, wherein the feeder cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

18. The method according to claim 8, wherein the feeder cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

The method according to claim 9, wherein the stem cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

20. The method according to claim 10, wherein the vegetative cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

The method according to claim 11, wherein the stem cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

22. The method according to claim 12, wherein the feeder cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

The method according to claim 13, wherein the feeder cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.

24. The method according to claim 14, wherein the stem cell into which the Wnt3a gene is introduced is a cell expressing Wnt3a.