KR100981093B1 - The development of human placenta mesenchymal stem cells to promote the proliferation of CD34+ hematopoietic stem cells and CD14+ monocyte from mononuclear cells from human bone marrow and cord blood - Google Patents

Abstract

The present invention supports for culturing mononuclear cells collected from human bone marrow or cord blood from mesenchymal stem cells purified and separated from human placenta, which can be collected from a normal mother without harm to a donor after normal delivery and without sufficient cost. The present invention relates to a technique for inducing proliferation culture of CD34 + hematopoietic stem cells and differentiation culture to CD14 + monocytes in a serum-free medium.

According to the present invention, CD34 + hematopoietic stem cells and CD14 + monocytes from human bone marrow and umbilical cord blood are used as human supportive cells for human cultured for production of CD34 + hematopoietic stem cells and CD14 + monocytes from human bone marrow and umbilical cord blood. Differentiated production can be usefully applied to the actual cell therapy.

Description

The development of human placenta mesenchymal stem cells to promote the proliferation of CD34 + hematopoietic stem cells and CD14 + monocyte from mononuclear cells using human placental mesenchymal stem cells to CD34 + hematopoietic stem cells or CD14 + monocytes from human bone marrow and cord blood}

The present invention provides human placenta as feeder cells in culture for proliferation production from mononuclear cells derived from human bone marrow and umbilical cord blood to CD34 + hematopoietic stem cells and differentiation production to CD14 + monocytes. The present invention relates to a technique for applying mesenchymal stem cells derived from human placenta. More specifically, the present invention comprises inoculating human bone marrow or cord blood mononuclear cells onto a support cell consisting of human placental mesenchymal stem cells, and culturing the inoculated human bone marrow or cord blood mononuclear cells by adding a serum-free animal cell culture medium. The present invention relates to a method for culturing differentiation from human bone marrow or cord blood mononuclear cells to CD34 + hematopoietic stem cells or CD14 + monocytes.

Another object of the present invention is to use human placental-derived mesenchymal stem cells as support cells in culture for proliferation production from human bone marrow and cord blood to CD34 + hematopoietic stem cells and differentiation into CD14 + monocytes. To develop culture technology for the production of CD34 + hematopoietic stem cells and CD14 + monocytes from human bone marrow and umbilical cord blood-derived monocytes by demonstrating that the proliferation and differentiation cultivation efficiency is comparable to or better than that of conventional human bone marrow-derived mesenchymal stem cells. have. The present technology of applying human placental mesenchymal stem cells can efficiently differentiate culture products of monocytes derived from human bone marrow and umbilical cord into CD34 + hematopoietic stem cells or CD14 + monocytes, which can be usefully applied to actual cell therapy.

Since human bone marrow and cord blood-derived mononuclear cells contain a large number of hematopoietic stem cells, they are currently attracting attention as an important research subject for the treatment of intractable disease. However, to date, application of supporting cells for culture is essential for the proliferation and differentiation of human bone marrow and cord blood-derived mononuclear cells. However, stromal cells derived from animals such as rats have been used as support cells for the present culture, which acts as a factor that makes the actual clinical application of culture products of human bone marrow and cord blood-derived mononuclear cells difficult. Therefore, the development of technology to apply human-derived stromal cells for the culture of human bone marrow and umbilical cord blood-derived mononuclear cells is required, the research and technology development related to this is being progressed worldwide.

Currently, CD34 + hematopoietic stem cell proliferation from human bone marrow and umbilical cord blood-derived monocytes and differentiation culture of CD14 + monocytes have been used in complex culture techniques with animal-derived stromal cells, such as mouse fibroblasts and cells that have established them as cell lines. Based on.

The complex culture technique with the animal-derived stromal cells is less likely to cause problems when used for research purposes, but the interaction between human bone marrow and umbilical cord blood-derived mononuclear cells and animal stromal cells for direct application to the human body. Unexpected problems such as production of genetically modified cells in the genus or invasion of animal-derived pathogens may occur.

In order to solve the problems existing in the prior art, there have been various studies to develop human-derived support cells such as bone marrow mesenchymal stem cells and subcutaneous fibroblasts, but the technology for commercialization has not yet been developed. same. First, the person supplying human-derived support cells should be a normal person, and should not cause serious problems to the provider during the collection process, the supply of human-derived support cells should be sufficient and smooth, and the cost-effectiveness of the collection should be appropriate. This is because it is difficult to satisfy these requirements. In addition, the biggest difficulty in commercialization is that the proliferation and culture effect of human embryonic stem cells should be comparable or superior to that of animal-derived support cells. A similar effect to animal-derived support cells was reported in 2004 by McNiece et al. (Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells.I McNiece, J Harrington, J Turney, J Kellner J, EJ Shapall.Cytotherapy, 2004 Vot 6 (No. 4), 311-317), but in this case, an invasive manipulation involving the risk of collecting bone marrow is required for normal bone marrow mesenchymal stem cell collection. There was a problem.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, when using human placenta-derived mesenchymal stem cells as support cells for the culture of human bone marrow and cord blood-derived mononuclear cells, derived from the existing human bone marrow When the mesenchymal stem cells were applied, the proliferation and differentiation culture efficiency was confirmed to be similar or very good, and the present invention was completed.

Accordingly, a main object of the present invention is to provide a method for separating and culturing mesenchymal stem cells from human placenta and using them as support cells to proliferate culture of CD34 + hematopoietic stem cells from human bone marrow and umbilical cord blood-derived mononuclear cells or to differentiate them into CD14 + monocytes. There is.

It is another object of the present invention to provide CD34 + hematopoietic stem cells or CD14 + monocytes using a differentiation method from human bone marrow and cord blood mononuclear cells using human placental mesenchymal stem cells to CD34 + hematopoietic stem cells or CD14 + monocytes.

Another object of the present invention is to separate and culture the mesenchymal stem cells from the human placenta, which can be collected without harm to the donor after normal delivery from the normal mother and without sufficient cost, and then used as support cells for human bone marrow and cord blood. Human bone marrow that can be applied to the human body more safely and efficiently by demonstrating that it is comparable to or superior to human bone marrow-derived mesenchymal stem cells that are effective in the proliferation of CD34 + hematopoietic stem cells from derived monocytes or the differentiation of CD14 + monocytes. Development of culture production technology of CD34 + hematopoietic stem cells and CD14 + monocytes from umbilical cord blood-derived monocytes.

To this end, Applicants et al. Attempted to produce CD34 + hematopoietic stem cells and CD14 + monocytes from human bone marrow and umbilical cord blood-derived mononuclear cells using human placental-derived mesenchymal stem cells as supporting cells. Comparison with cultured human bone marrow and cord blood-derived mononuclear cells was confirmed by colony culture and microscopic observation.

According to an aspect of the present invention, the present invention comprises inoculating human bone marrow or cord blood mononuclear cells onto support cells consisting of human placental mesenchymal stem cells, and adding the serum-free animal cell culture to the inoculated human bone marrow or cord blood mononuclear cells. Provided is a method for differentiating culture from human bone marrow or cord blood mononuclear cells to hematopoietic stem cells comprising culturing.

In the method for differentiation of human bone marrow or cord blood mononuclear cells to hematopoietic stem cells of the present invention, the human placental mesenchymal stem cells are collected from placental chorion cells after delivery and cultured in DMEM culture, where the grown placenta cells are grown. It is preferred that the cells were passaged by trypsin-EDTA and the cells passaged were irradiated with 1500 cGy of radiation.

According to another aspect of the present invention, the present invention comprises inoculating human bone marrow or cord blood mononuclear cells onto support cells consisting of human placental mesenchymal stem cells, inoculating human bone marrow or cord blood mononuclear cells with F-ligand and interleukin-7. Provided is a method for differentiating culture from human bone marrow or cord blood mononuclear cells to monocytes, comprising culturing by adding the serum-free animal cell culture solution.

In the method for differentiation of human bone marrow or umbilical cord mononuclear cells to monocytes of the present invention, the human placental mesenchymal stem cells are collected from placental chorion cells after maternal delivery and cultured in DMEM culture, where the grown placenta cells are grown. It is preferred that the cells were passaged by trypsin-EDTA and the cells passaged were irradiated with 1500 cGy of radiation.

According to another aspect of the present invention, the present invention comprises inoculating human bone marrow or cord blood mononuclear cells onto support cells consisting of human placental mesenchymal stem cells, and adding the serum-free animal cell culture to the inoculated human bone marrow or cord blood mononuclear cells. It provides a hematopoietic stem cell prepared by the method of differentiation culture from human bone marrow or cord blood mononuclear cells to hematopoietic stem cells, comprising the step of culturing.

According to another aspect of the present invention, the present invention comprises inoculating human bone marrow or cord blood mononuclear cells onto support cells consisting of human placental mesenchymal stem cells, inoculating human bone marrow or cord blood mononuclear cells with F-ligand and interleukin-7. Provided is a monocyte prepared by a method for differentiation of human bone marrow or cord blood mononuclear cells to monocytes, comprising culturing by adding the serum-free animal cell culture solution.

In the method of the present invention, serum-free animal cell culture is used. The serum-free culture medium used herein does not contain serum derived from animals, including humans, and means a culture medium for animal cell culture composed of chemically identified substances. The culture medium to which the purified and identified substance which comprises a serum is added is contained in the serum-free animal cell culture solution which concerns on this invention. Basic components of such cultures include amino acids, sugars, energy sources such as organic acids, vitamins, pH adjusting buffers, inorganic salts, and the like. The culture may contain a pH indicator such as phenolic. Serum-free animal cell cultures include, but are not limited to, RPMI cultures, Iscove's cultures, minimum essential cultures, Dulbeccos Modified Eagles Medium, and others known in the art (see, for example, US Pat. 6,733,746). Commercially available basic cultures include, but are not limited to, Stemline ^™ (Sigma Aldrich), StemSpan ^™ (StemCell Technologies, Vancouver, Canada), Stempro ^™ (Life Technologies, Gibco BRL, Gaithersburg, MD, USA) HPGM ^™ (Cambrex, Walkersville, MD, USA ), QBSF TM (Quality Biological, Gaithersburg, MD, USA), X-VIVO (Cambrex Corp., Walkersville, MD, USA) and ^{Mesencult TM (Stem Cell Technologies, Vancouver} , Canada) is Included. Formulations of these and other cultures will be apparent to those skilled in the art.

The present technology consists of two steps, including purification of human placental mesenchymal stem cells and their application to the growth of CD34 + hematopoietic stem cells from human bone marrow and umbilical cord blood-derived mononuclear cells and to the differentiation culture to CD14 + monocytes.

First, the purification process of human placental mesenchymal stem cells applied as support cells to promote the proliferation of human bone marrow and cord blood-derived mononuclear cells from CDN + hematopoietic stem cells or the differentiation culture of CD14 + monocytes from the placenta collected after normal delivery is as follows. Is the same as Stem cells used as human-derived support cells were collected chorion cells from the placenta after delivery and washed three times in dPBS, and the tissues chopped finely with surgical scissors were placed in a 25m ² flask, and the DMEM culture was changed once every two days. Incubate for a week. Placental cells grown in this tissue were treated with Trypsin-EDTA and cultured in a new flask for passage. Placenta cells thus prepared were used as culture support cells after 1500 cGy irradiation with 137 Cs gamma-irradiator, and the rest were frozen and stored for later use by increasing the number of cells after passage. The irradiation treatment of 1500 cGy for the preparation of human placental-derived mesenchymal stem cells takes place one day before coculture. In the case of irradiating the supporting cells without chemical treatment, human placenta-derived mesenchymal stem cells can be prepared under uniform conditions, unlike the chemical treatment method with slight fluctuations depending on the experimenter. Place each cell in a 35mm culture dish coated with 0.1% gelatin for 30 minutes and replace the culture immediately before transfer the next day. Adipocyte differentiation and osteoblast differentiation cultures are performed to confirm that human placenta-derived mesenchymal stem cells are correctly purified and isolated. This patent is the first example of irradiation of 1500 cGy in order to use human placental-derived mesenchymal stem cells as a culture supporting cell of human mononuclear cells. At this time, the overall microscopic appearance of the produced human placenta-derived mesenchymal stem cells (Fig. 1) and the differentiation ability into adipocytes and bone cells can be observed in the same manner as the human bone marrow-derived mesenchymal stem cells. In addition, it can be confirmed that the chromosome test has a normal chromosome without mutation (FIG. 4).

The purification process of human bone marrow and cord blood-derived monocytes is as follows. Umbilical cord blood is collected and stored frozen before delivery to placenta, and known methods such as Ficoll-Hypaque density gradient method can be used to separate monocytes from umbilical cord blood. Specifically, the bone marrow or umbilical cord blood is diluted with phosphate buffered saline (PBS), diluted, and then superimposed on the same amount of Ficoll-Hypaque (Ficoll-Hypaque, density; 1.077 g / ml) with the diluted bone marrow or umbilical cord blood. Let's do it. At this time, it is preferable that the Ficoll-Hippack solution is brought to room temperature before use, and that the volume of diluted bone marrow or umbilical cord blood does not exceed three times the volume of the Ficol-Hippack solution. It is centrifuged to separate the erythrocyte layer, monocyte layer and serum layer. Pasteur pipettes (pasteur pipette) to transfer the mononuclear cell layer only to a new tube, and washed with PBS, etc. to remove the collected Picol-Hippack solution or contaminated platelets. The mononuclear cells thus separated can be used immediately for isolation and culture of multipotent progenitor / stem cells or can be used after prolonged storage by cryogenic freezing.

The culture technique using CD34 + hematopoietic stem cell proliferation production from mononuclear cells of bone marrow and umbilical cord using human placenta-derived mesenchymal stem cells produced by the aforementioned method as the supporting cells is as follows. First, human placental mesenchymal stem cells irradiated with 1500 cGy one day before co-culture were incubated in a T75 container at 2.0 X 10 ⁵ for 24 hours to confirm that they were attached to the culture vessel surface. The cells were cultured for 4 weeks using 1.0 cc 10 ⁶ of QBSF serum free media (Sigma Aldrich, USA) as a culture medium for 1.0 × 10 ⁶ mononuclear cells isolated from bone marrow and cord blood by Ficoll-Hypaque technique. Cultures are collected for cell counting and analysis by half of the total volume every week, fresh cultures are added and the procedure lasts 4 weeks. At this time, using the bone marrow-derived mesenchymal stem cells produced by the same technique as the placental-derived mesenchymal stem cells as support cells, by measuring the culture efficiency of the bone marrow and cord blood-derived mononuclear cells cultured through the same process, Compare and analyze those of stem cells. The control group is a case in which bone marrow and cord blood-derived mononuclear cells were cultured only in the same medium as described above without co-culture with support cells. As a result, bone marrow and cord blood mononuclear cells cultured with human placenta-derived mesenchymal stem cells as supporting cells between 1-4 weeks of culture were not significantly different from those of human bone marrow-derived mesenchymal stem cells. Very significantly, it was observed that there is a culture growth effect of CD34 + cells. The corresponding results are shown in FIG. 5 showing the proliferative effect in each culture state and FIG. 6 showing the flow cytometry results. In order to confirm that the cells were true CD34 + hematopoietic stem cells, colony culture was performed in Methocult semisolid Cellulose medium. As a result, it was confirmed that CFU-GEEM and CFU-GM proliferated and differentiated from hematopoietic stem cells were observed. Prove that hematopoietic stem cells (Fig. 7). This technique is characterized by an attempt to proliferate and culture CD34 + hematopoietic stem cells without using cytokines that promote cell growth in serum-free culture.

In addition, QBSF mixed with F-ligand and Interleukin-7 at a concentration of 20 ng / mL was used to promote differentiation of CD14 + monocytes from bone marrow and cord blood-derived monocytes using human placenta-derived mesenchymal stem cells as support cells. 20 cc of serum free media (Sigma Aldrich, USA) was used as a culture medium, and cultured for 24 hours in a T75 container attached with 2.0 x 10 ⁵ human placental mesenchymal stem cells irradiated at 1500 cGy one day before coculture. Incubate 1.0 x 10 ⁶ mononuclear cells in a T75 culture vessel with human placental mesenchymal stem cells attached to the culture vessel surface. The incubation lasts six weeks. Other cytokine mixtures were compared with adenoviruses containing Thrombopoietin-producing genes, 50 mg / mL each of K-ligand, F-ligand, and Thrombopoietin, and no cytokines. As a result, it was observed that CD14 + monocytes had differentiation effect in F-ligand and Interleukin-7 groups. Corresponding results are shown in FIG. 8 showing the CD14 + monocyte differentiation effect and FIG. 9 showing the flow cytometry results. And microscopic observation to confirm that these cells are true CD14 + monocytes was able to confirm the appearance of typical monocytes (Fig. 10). The technique is characterized by an attempt to differentiate and culture CD14 + monocytes using cytokines that promote cell growth in serum free cultures.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  1.Derived from human bone marrow and cord blood Monocytes  Human placenta acts as a support cell in culture Mesenchyme  Stem cell production and culture technology

In the present invention, the following experiments were performed to prepare human placental mesenchymal stem cells serving as support cells in the culture of human bone marrow and cord blood-derived mononuclear cells. The following experiment was repeated a total of 10 times.

1-1. Isolation of Human Placental Mesenchymal Stem Cells

In order to isolate human placental mesenchymal stem cells, chorion tissue was collected from the placenta after delivery. It was washed three times with sterile saline solution, chopped enough with sterile surgical scissors and placed in a 25 cm ² flask. Here, DMEM (Dulbecco's Modified Eagle Medium, Gibco Co., Ltd.) culture medium was replaced once every two days and incubated for one week. Mesenchymal stem cells grown in the cultured tissues were treated with Trypsin-EDTA (10 ml), and then transferred to a new flask for subculture and cultured. Human placenta mesenchymal stem cells will be present when three to four passages are performed. Flow cytometry (Becyton-Dickinson, USA) was performed on the isolated mesenchymal stem cells, and separation and purification confirmation experiments were performed as described below.

1-2. Isolation and Purification of Human Placental Mesenchymal Stem Cells

When the isolated human placental mesenchymal stem cells were used as support cells for culturing human bone marrow and umbilical cord blood-derived mononuclear cells, 1,500 cGy of radiation was used as culture support cells. Stretched and stored frozen for later use. Preparation of human placental mesenchymal stem cells as support cells was performed one day before the culture of bone marrow and cord blood-derived mononuclear cells. Each cell was plated in a 35 mm dish coated with 0.1% gelatin for 30 minutes, and the medium was replaced with the same amount of MEM (Dulbecco's Modified Eagle Medium, Gibco) immediately before transfer. Direct microscopic observation (FIG. 1) and isolated human placental mesenchymal stem cells were FITC-labeled anti-CD44, CD73, CD105 and CD29 antibodies (Becton-) to confirm that human placental mesenchymal stem cells were correctly purified and isolated. Dickinson's staining), the fluorescence intensity (Fluorescence intensity) was measured by flow cytometry, and the differentiation culture into adipocytes (Fig. 2) and bone cells (Fig. 3).

Chromosome karytype analysis was performed to confirm stability of chromosomes in the complex culture of human placental stromal cells of the present invention. As a result, human placental stromal cells showed normal 46XY findings up to 50 passages (FIG. 4).

Example 2 Production of CD34 + Hematopoietic Stem Cells from Human Bone Marrow and Umbilical Cord Blood-Derived Monocytes Using Human Placental Mesenchymal Stem Cells as Supporting Cells

2-1. Purification of Human Bone Marrow and Umbilical Cord Blood-Derived Monocytes

The purification process of human bone marrow and cord blood-derived monocytes is as follows. Umbilical cord blood is collected and stored frozen before delivery to placenta, and known methods such as Ficoll-Hypaque density gradient method can be used to separate monocytes from umbilical cord blood. Specifically, the bone marrow or umbilical cord blood is diluted with phosphate buffered saline (PBS), diluted, and then superimposed on the same amount of Ficoll-Hypaque (Ficoll-Hypaque, density; 1.077 g / ml) with the diluted bone marrow or umbilical cord blood. Let's do it. At this time, it is preferable that the Ficoll-Hippack solution is brought to room temperature before use, and that the volume of diluted bone marrow or umbilical cord blood does not exceed three times the volume of the Ficol-Hippack solution. It is centrifuged to separate the erythrocyte layer, monocyte layer and serum layer. Pasteur pipettes (pasteur pipette) to transfer the mononuclear cell layer only to a new tube, and washed with PBS, etc. to remove the collected Picol-Hippack solution or contaminated platelets. The mononuclear cells thus separated can be used immediately for isolation and culture of multipotent progenitor / stem cells or can be used after prolonged storage by cryogenic freezing.

2-2. Production of CD34 + Hematopoietic Stem Cells from Human Bone Marrow and Umbilical Cord Blood Derived Monocytes

In order to demonstrate that CD34 + hematopoietic stem cells can be produced from human bone marrow and cord blood-derived mononuclear cells using human placental mesenchymal stem cells, which are the core of the present invention, five human bone marrow-derived mononuclear cells and five cases 10 human placental-derived mesenchymal stem cells were applied to human umbilical cord blood-derived mononuclear cells as culture support cells and analyzed for production efficiency of CD34 + hematopoietic stem cells. At this time, 20 cc of O ₂ concentration, 5% of CO ₂ concentration, and 37 ^° C. were cultured using 20 cc of QBSF serum free media (Sigma Aldrich, USA) as a culture medium. The experiment was repeated 10 times in total.

As a result, the production efficiency of CD34 + hematopoietic stem cells was 13.6 ± 5.3 for placental support cell group, 14.7 ± 3.8 for bone marrow support cell group (American Type Culture Collection), 5.1 for control group (control group without supporting cells). The number of CD34 + hematopoietic stem cells of ± 2.3 was shown (FIG. 5). At 2 weeks of culture, the number of CD34 + hematopoietic stem cells was 29.7 ± 8.3 in the placental support cell group, 27.9 ± 7.4 in the bone marrow support cell group, and 6.7 ± 3.7 in the control group. The peak of the support cell group was 41.0 ± 7.2 and the control group was 6.9 ± 4.7. At 4 weeks of culture, the placental support cell group, the bone marrow support cell group, and the control group showed deterioration.

Statistical analysis showed that when human placental mesenchymal stem cells were used as support cells, they showed the same CD34 + hematopoietic stem cell proliferation effect as those using human bone marrow mesenchymal stem cells. <0.001).

CD34 + hematopoietic stem cells were stained with anti-CD34 antibody (Becton-Dickinson) and anti-CD38 antibody (Becton-Dickinson) labeled with 5'-fluorescein isothiocyanate (FITC) and Phycoerythrin (PE), and flow cytometry, Fluorescence intensity was measured using Becton-Dickinson, USA. Flow cytometry revealed that most of the proliferated CD34 + cells were CD34 + / 38 + hematopoietic stem cells in the differentiation process. At the 3rd week of culture, the proportion of CD34 + / 38 + in the total CD34 + cells was 96.9 ± 5.7% in the placental support cell group, 98.5 ± 11.3% in the bone marrow support cell group, and 92.7 ± 9.3% in the control group. 6). In addition, CFU-GEMM (colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte), and CFU-GM (colony forming) corresponding to progenitors were performed as a result of Methocult Semisolid culture (MethoCult, Stem Cell Technologies, Vancouver) performed on the cells. unit-granulocyte, macrophage) and the like, it was confirmed that it has a function as CD34 + hematopoietic stem cells (Fig. 7).

Example  3. Human Placenta Mesenchymal stem cells  As a supporting cell Human bone marrow  And cord blood From mononuclear cells CD14 + Production technology of monocytes

3-1. Derived from human bone marrow and umbilical cord blood Mononuclear  refine

Human bone marrow and umbilical cord blood-derived mononuclear cells were purified in the same manner as in Example 2-1.

3-2. human Bone marrow and  Cord blood origin From monocytes CD14 + Production of monocytes

In order to demonstrate the production of CD14 + monocytes from human bone marrow and umbilical cord blood-derived monocytes using human placental mesenchymal stem cells of the present invention, five human bone marrow-derived monocytes and five human cord blood-derived monocytes were identified. A total of 10 human placenta-derived mesenchymal stem cells were used as culture support cells to compare the production efficiency of CD14 + hematopoietic stem cells in various cytokine combinations. 20 cc of QBSF serum free media (Sigma Aldrich, USA) was analyzed. The culture medium was incubated at a temperature of 20% O ₂ , 5% CO ₂ and 37 ^° C. The experiment was repeated 10 times in total.

The experiment was divided into a total of six groups, the experiment was carried out according to the conditions described in Table 1 below.

TABLE 1

Cytokines Used Culture medium exchange Incubation period Placental mesenchymal cell irradiation (1,500 cGy_) Adeno-TPO group 200 PFU * / mL Every week 6 Weeks Irradiation FL / IL-7 Military 20 ug / mL Every week 6 Weeks Irradiation KL / FL / TPO County 50 ug / mL Every week 6 Weeks Irradiation Adeno-TPO group 200 PFU / mL Every week 6 Weeks No irradiation FL / IL-7 Military 20 ug / mL Every week 6 Weeks No irradiation KL / FL / TPO County 50 ug / mL Every week 6 Weeks No irradiation

* PFU: Plaque Forming Unit

As a result, the production efficiency of CD14 + monocytes was 430,000 / uL in the F-ligand and Interleukin-7 groups at the 6th week of culture. (FIG. 8) This is a very significant result even by statistical analysis (p <0.001). In addition, the production efficiency of CD14 + monocytes was increased in the radiation group than in the non-irradiation group.

Mononuclear cells were labeled with anti-CD14 antibody (Becton-Dickinson) and anti-CD34 antibody (Becton-Dickinson) labeled with Allophycocyanin (PEC) and Phycoerythrin (PE), and fluorescence intensity was determined by flow cytometry (FACS Calibur. BD bioscience). Fluorescence intensity was measured. As a result of flow cytometry, it was confirmed that almost no CD34 + cells were proliferated except the differentiated CD14 + cells. After monocytes were isolated from the cultured cells for this experiment, 20uL of anti-CD14 and CD34 monoclonal antibodies (anti-HPCA-2, Becton-Dickinson) with APC and PE were added, followed by reaction at 4 ° C for 25 minutes in the dark. I was. This was once washed twice at 300 g for 7 minutes each with 1 mL and 2 mL of 0.1% sodium azide (Sigma) and 2% fetal bovine serum, and then suspended in 1 mL of wash solution and FACS scan (Becton-Dickinson, USA) The level of CD14 + CD34 − cells was measured using.

Therefore, it was proved that the present culture was a selective culture that differentiated into CD14 + monocytes rather than the proliferation culture of CD34 + hematopoietic stem cells (FIG. 9). In addition, the microscopic observation of the cells showed that they were typical immune monocytes. (Figure 10)

As described above, according to the present invention, normal human placental mesenchymal stem cells which can be safely supplied to infinity can be used as support cells for proliferation from human bone marrow and cord blood-derived mononuclear cells to CD34 + hematopoietic stem cells, It can be seen that can be used as a support cell for differentiation from cord blood-derived monocytes to CD14 + monocytes.

In addition, the present technology of applying human placental mesenchymal stem cells for the production of CD34 + hematopoietic stem cells and CD14 + monocytes from human bone marrow and umbilical cord blood-derived monocytes can differentiate and produce CD34 + hematopoietic stem cells and CD14 + monocytes from human bone marrow and umbilical cord blood. It can be usefully applied to cell therapy.

1 is a view of purified, isolated human placental mesenchymal stem cells (No Stain, X 40).

Figure 2 shows the appearance of adipocytes differentiated from human placental mesenchymal stem cells (No Stain, X 40).

Figure 3 is a bone cell differentiation from human placental mesenchymal stem cells (Von Kossa Stain, X 40).

4 shows that chromosomes of isolated human placental mesenchymal stem cells are normal without mutation.

5 is a graph showing that the proliferative effect of CD34 + hematopoietic stem cells from human mononuclear cells is not different from that of human placental mesenchymal stem cells.

FIG. 6 shows flow cytometry indicating that most CD34 + / 38 + cells are used when human placental mesenchymal stem cells are used as support cells, when human bone marrow mesenchymal stem cells are used as support cells, and in controls cultured without support cells. ) (A) human placental mesenchymal stem cells, (b) human bone marrow mesenchymal stem cells, (c) control cells not used.

7 is a Methocult CFU-GEEM colony culture of differentiation-induced CD34 + hematopoietic stem cells using human placental mesenchymal stem cells (X 200).

8 shows that human mononuclear cells using F-ligand and Interleukin-7 when inducing differentiation from human bone marrow or umbilical cord blood-derived mononuclear cells into CD14 + monocytes using human placental mesenchymal stem cells as support cells were significantly more than those using other cytokines. From CD14 + monocytes.

9 is a flow cytometry analysis showing that the differentiation effect of CD14 + monocytes from human monocytes occurs selectively without proliferation of CD34 + cells.

Figure 10 shows the appearance of CD14 + monocytes cultured using human placental mesenchymal stem cells (Wright Stain, X 400).

Claims (6)

Human bone marrow or umbilical cord blood mononuclear cells were collected after maternal chorionic villus cells were cultured and cultured in DMEM medium, and the placental cells grown by trypsin-EDTA were passaged and irradiated to the passaged cells. Differentiation from human bone marrow or cord blood mononuclear cells to hematopoietic stem cells, comprising inoculating onto support cells consisting of placental mesenchymal stem cells, and culturing the inoculated human bone marrow or cord blood mononuclear cells with a serum-free animal cell culture medium Culture method. delete Human bone marrow or umbilical cord blood mononuclear cells are obtained after maternal chorionic villus cells are harvested and cultured in DMEM medium, and the grown placenta cells are passaged by trypsin-EDTA treatment and irradiated with passaged cells. Inoculating onto a support cell consisting of human placental mesenchymal stem cells, and culturing the inoculated human bone marrow or cord blood mononuclear cells by adding a serum-free animal cell culture solution containing F-ligand and interleukin-7, Method for Differentiation of Human Bone Marrow or Umbilical Cord Blood Monocytes to Monocytes. delete delete delete

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