KR20170084495A

KR20170084495A - Method of simultaneously differentiation of stem cells derived endothelial cell and hemangioblast without sorting

Info

Publication number: KR20170084495A
Application number: KR1020160003591A
Authority: KR
Inventors: 문성환; 정형민; 길창현
Original assignee: 건국대학교 글로컬산학협력단
Priority date: 2016-01-12
Filing date: 2016-01-12
Publication date: 2017-07-20
Also published as: WO2017122887A1

Abstract

The present invention relates to a method for simultaneously differentiating stem cell-derived vascular cells and hematopoietic stem cells, comprising the steps of treating stem cells with DPBS to form single cells; Floating the single cells to form aggregates; Classifying the aggregate into an angiogenic differentiation group and a hematopoietic differentiation group; And differentiating the two groups of aggregates.
Also, according to the present invention, since purification process is required to select a desired cell in the existing cell differentiation process, a separate purification process is not required, and thus, cost and time can be saved.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for simultaneous differentiation of stem cell-derived vascular cells and hematopoietic stem cells,

More particularly, the present invention relates to a method for simultaneous differentiation of stem cell-derived vascular cells and hematopoietic stem cells, and more particularly, to a method for the simultaneous differentiation of embryonic stem cell clusters into single cells using DPBS, Without purification process.

Stem cells are cells that are capable of differentiating into various types of cells and are used to grow into an alternative tissue that has the ability to grow itself as one of several cell types present in the body It has been used and studied for the treatment of various diseases.

Among them, human embryonic stem cells are cells derived from the inner cell mass of human blastocysts and have self-proliferation and differentiation ability. Methods for inducing differentiation into specific cells using human embryonic stem cells have been used to induce differentiation into specific cells through an embryoid body stage characterized by triploidity or to treat specific cytokines .

The development of endothelial cells is differentiated from hemangioblasts and angioblasts, which are mesodermal cells in the triple lobe, and they form blood vessels through vasculogenesis and angiogenesis.

The process by which blood vessels are formed in the human body are largely divided into two types: an angiogenesis mechanism in which vascular endothelial cells are moved from surrounding blood vessels into a surrounding tissue, and a vascularization mechanism in which circulating cells in the blood return to tissues to form blood vessels Path. In 1997, it was discovered that blood vessels were formed by vascular endothelial progenitor cells by Asahara of Elizabeth Medical Center of Tufts University.

Vascular cells play an important role in angiogenesis and are involved in the maintenance of all tissues in the body and in the regulation of neovascularization, inflammation and thrombosis. In order to produce such vascular cells, human embryonic stem cells in an undifferentiated state are induced into an embryo, immunoreacted with an antibody expressed in vascular cells, and separated by a method using laser (FACS) and magnets (MACS) But there was a limit in that the method was difficult, and the time and expense was large.

Korean Patent No. 10-0948170 (Mar. 10, 2010) and Korean Patent Laid-Open No. 10-2013-0105124 (Mar.

The present inventors devised a cell differentiation method mimicking the in vivo cell differentiation process of a human, and obtained blood cells and vascular cells simultaneously according to a cell differentiation method using DPBS, thereby completing the present invention.

It is an object of the present invention to provide a method and apparatus for simultaneously differentiating hematopoietic cells and vascular cells using a method designed to mimic human cell differentiation processes to more easily perform differentiation of hematopoietic cells and blood vessel cells, To provide a way to do that.

In order to accomplish the above object, the present invention provides a method for treating a stem cell, And floating the single cells to form an aggregate. The present invention also provides a method for differentiating stem cell-derived vascular cells from untreated cells.

The present invention further provides a method for differentiating stem cell-derived vascular endothelial cells from the above method or an embodiment, further comprising the step of dissociating and co-culturing the aggregate.

In one embodiment of the present invention, the adherent culture may be performed in a collagen culture medium.

In one embodiment of the present invention, the stem cells may be selected from the group including embryonic stem cells, induced pluripotent stem cells, somatic cell cloning stem cells, and pluripotent stem cells.

(1) treating stem cells with DPBS to form single cells; (2) suspending the single cells to form aggregates; (3) classifying the aggregate of step (2) into an angiogenic differentiation group and a hematopoietic differentiation group; And (4) dividing the aggregates of the two groups classified by the step (3) into a stem cell-derived vascular cell and a hematopoietic stem cell differentiation.

In one embodiment of the present invention, in the step (4), the step of differentiating the aggregated body of the vascular cell differentiation group may be a step of dissociating and attaching the aggregated body.

In one embodiment of the present invention, in the step (4), the step of differentiating the agglutinating group of the hematopoietic differentiation group may be a step of suspending the aggregate.

According to the present invention as described above, the embryoid body is single-celled using DPBS in stem cells, and then the suspension is cultured to form an aggregate, whereby the aggregate is monoclonalized to differentiate or vascularize the blood vessel cells, It is possible to simultaneously differentiate the blood vessel cells and the blood stem cells simultaneously.

Also, according to the present invention, since purification process is required to select a desired cell in the existing cell differentiation process, a separate purification process is not required, and thus, cost and time can be saved.

Brief Description of the Drawings Fig. 1 is a diagram showing the process of differentiation of vascular cells and blood stem cells according to an embodiment of the present invention, (A) the progression by day, (B) the process of embryoid body formation from stem cells, (C) And (D) a schematic diagram showing a process of forming blood stem cells using an embryoid body.
FIG. 2 is a photograph showing the characteristics of differentiated blood vessel cells according to an embodiment of the present invention as an expression amount of a gene through RT-PCR. FIG.
FIG. 3 is a photograph showing the expression of vascular cell-specific protein vWF and PECAM differentiated according to an embodiment of the present invention through immunocytochemistry.
FIG. 4 is a photograph showing the expression levels of differentiated vascular cell-specific proteins (A) SSEA-4, (B) Tie-2, and (C) PECAM according to an embodiment of the present invention through fluorescence activated flow cytometry.
FIG. 5 is a photograph showing the tube structure-forming ability by confirming the expression of differentiated vascular cell-specific protein (A) PECAM, (B) vWF and (C) VE-cadherin according to an embodiment of the present invention.
FIG. 6 is a photograph showing the expression level of differentiated vascular cell-specific Ac-LDL according to an embodiment of the present invention in hESC-EC and hCB-EPC.
FIG. 7 is a photograph and a table comparing the symptoms of a lower limb ischemic rat model injected with differentiated blood vessel cells according to an embodiment of the present invention with a control group. FIG.
FIG. 8 is a photograph (A) and a graph (B) showing the therapeutic effect of a subcutaneous ischemic rat model injected with differentiated blood vessel cells according to an embodiment of the present invention, using a Doppler laser apparatus.
9 is a micro CT image showing the therapeutic effect of a subcutaneous ischemic rat model injected with differentiated blood vessel cells according to an embodiment of the present invention.
FIG. 10 is a photograph (A) and a graph (B) in which the treatment effect of a subcutaneous ischemic rat model injected with differentiated blood vessel cells according to an embodiment of the present invention was analyzed by tissue immunoassay.
FIG. 11 is a photograph showing a therapeutic effect of a subcutaneous ischemic rat model injected with differentiated blood vessel cells according to an embodiment of the present invention through fluorescence in situ examination. FIG.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for treating a stem cell by treating Dulbecco's phosphate buffered saline (DPBS) And then suspending the single cells to form an aggregate. The present invention also provides a method for differentiating stem cells into endothelial cells.

Floating culture is a culture method in which the cells are suspended in a culture solution when the animal cells are cultured. Cells that proliferate in the suspended state, such as blood cells and ascites cancer cells, can be suspended in culture even if they are not shaken or rotated. However, in most cases, the cells are rotated or cultured (shake culture) ), Or it may be necessary to rotate the incubator (turn culture).

The present invention further provides a method for differentiating stem cell-derived vascular endothelial cells from the above-described method or one embodiment, further comprising the step of dissociating and coadministering the aggregate.

Adhesive culture is a culture method in which cells are adhered on a solid (agar) medium by a method applied to general animal cells, and induced to grow as a monolayer. This is also referred to as monolayer culture.

In one embodiment of the invention, the attachment culture can be performed in collagen medium.

In one embodiment of the present invention, the stem cells are selected from the group consisting of embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), somatic cell nuclear transfer (SCNT) And pluripotent stem cells (PSC).

(1) treating stem cells with DPBS to form single cells; (2) suspending the single cells to form aggregates; (3) classifying the aggregate of step (2) into an angiogenic differentiation group and a hemangioblast differentiation group; And (4) dividing the aggregates of the two groups classified by the step (3) into a stem cell-derived vascular cell and a hematopoietic stem cell differentiation.

When enzyme treatment is used for a single dissociation of cells, cell death or damage occurs in many cells. In the case of treating DPBS, it was confirmed that the cell differentiation could be progressed efficiently because the cell death was not induced and thus the cell survival rate could be improved.

Preferably, the DPBS according to the present invention does not contain calcium and magnesium. The use of DPBS containing calcium and magnesium for dissociation of cells results in decreased dissociation of cells.

In one embodiment of the present invention, the stem cells may be any one selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, somatic embryonic stem cells, and pluripotent stem cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Culture of human embryonic stem cells

Human embryonic stem cells were cultured in DMEM / F12, 1% MEM-NEAA, 0.1% beta-mercaptoethanol, 1% penicillin-streptomycin and 4 ng / ml bFGF. H9-hESCs (Wicell Research Institute, Madison, Wisconsin) were used as human embryonic stem cell cell lines. Human embryonic stem cells were co-cultured with mouse embryonic fibroblast (MEF) in which cell division was inactivated as nutrient cells, and subcultured every 5 to 7 days.

Example 2. Aggregation Formation Using Stem Cells

The cell differentiation process of the present invention disassembles into a single cell after formation of an embryoid body, and then forms an aggregate and simultaneously differentiates into two cells (FIG. 1A).

Human embryonic stem cells were cultured for 5-7 days to form embryoid bodies and then fibroblasts were removed. After the culture medium was removed, DPBS without calcium chloride and magnesium was added to dissociate into single cells.

More specifically, the cells were washed once with DPBS, 5 ml of DPBS was added, and the cells were incubated for 5 to 10 minutes under a CO ₂ atmosphere. The disruption of stem cell clusters was observed under a microscope and pipetting was carried out so that there was no damage to the cells.

Then, when the human embryonic stem cell clusters were disassociated into single cells, they were separated into pure single cells after being placed in a 40-μm mesh. The supernatant was removed by centrifugation at a speed of 1000 to 1500 rpm.

Low-attach dish (Stemcell) was prepared by adding Stemline II medium (Sigma-Aldrich), VEGF (Vascular endothelial growth factor, Peprotech) 50 ng / ml and BPP4 50 ng / For 2 days to form aggregates.

After 2 days, 50% of the culture was removed and 50% of the removed Stemline II culture medium and 50 ng / ml of VEGF, 50 ng / ml of BMP4, 50 ng / ml of TPO, 50 ng / ml of Flt- / ml, and 50 ng of bFGF. After further incubation for 2 days, aggregates were harvested (Fig. 1B).

Example 3 Differentiation of Vascular Cells Using Aggregates

The aggregates prepared in Example 2 were dissociated into single cells using 0.05% Trypsin-EDTA. Collagen type IV was coated on a 60 mm plate, and 5 x 10 ⁵ cells of the dissociated single cells were added. When the cell density in the collagen-coated plate was 80 to 90% or more, the cells were subcultured to produce human embryonic stem cell-derived vascular cells (Fig. 1C).

Example 4 Differentiation of Blood Blast Cells Using Aggregates

To prepare the aggregates prepared in Example 2 with hematopoietic stem cells, methylcellulose H4436 (methylcellulose) containing additives such as BMP4, SCF, VEGF, TPO, Flt-3, bFGF and EX-CYTE (Millipore, Billerica, MA) ; Stemcell Technologies, Vancouver, British Columbia, Canada) at a concentration of 5 × 10 ⁵ cells per well in a 6-well dish. After 2-3 days, grape clusters characteristic of hematopoietic cells appeared, and the differentiation was completed after 6 to 7 days (Fig. 1D).

Measurement example 1. Analysis of expression level of vascular cell-specific marker gene by RT-PCR

Via the ^{Trizol ® (Invitrogen, Carlsbad, CA} ) NanoDrop ® ND-1000 spectro photometer (NanoDrop Technologies, Wilmington, DE) using the order to analyze the expression level of the RNA was extracted for total RNA. First, reverse transcription was performed using Maxim RT pre-mix kit (iNtRon biotechnology, Sungnam, Korea) to obtain cDNA. Analysis by reverse transcription polymerase chain reaction (RT-PCR) was performed on undifferentiated H9-hESCs, hESC-ECs and CB-EPCs. AccuPower PCR premix (Bioneer, Daejeon, South Korea) was used and the cycle was set to repeat 35 cycles at 95 ° C for 30 seconds, at 55-60 ° C for 30 seconds, and at 72 ° C for 30 seconds. The PCR cycle started with an initial denaturation step of 5 min, followed by a 95 ° C, finalization with a final dilatation step of 10 min, proceeding to 72 ° C. The RT-PCR primer sequences are listed in Table 1. RT-PCR results were stained with EtBr (ethidium bromide) and confirmed by 1.5% agarose gel (Promega, Madison, Wis.) (Fig. 2).

RT-PCR was performed to confirm that the differentiation was induced by decreased expression of genes (oct-4 and nanog) that exhibited pluripotency and consequently increased expression of endothelial markers. Expression of hESCs - expressing genes was strongly expressed up to day 2 hEBs but not in hESC - derived ECs. Similar to control, CB-EPC, the induced EC resulted in endothelial progenitor cell (EPC) markers flt-1, tie-2, VE-cad, vwf and kdr (kinase insert domain receptor ) Was expressed.

Measurement example 2. Expression of vascular cell-specific protein by immunocytochemical analysis

Cells were fixed with 4% paraformaldehyde and treated with PBS (phosphate buffered saline; Sigma-Aldrich) containing 0.03% Triton X-100 for 5 minutes to increase permeability. After 5% normal goat serum was treated for 30 minutes, the cells were incubated at 4 ° C in an over-night condition with a primary antibody, PECAM (anti-human platelet endothelial cell adhesion molecule (BD Bioscience) and anti-human vanillin factor (vWF) BD Bioscience). in order to visualize the vWF and PECAM staining the cells were washed twice with Alexa Fluor ^® 488 and 594 secondary antibody (Molecular Probes Inc., Sunnyvale, CA ). All images were obtained using a fluorescence microscope (Nikon, Eclipse Ti, Chiyoda-ku, Japan). Finally, the nucleus was visualized by staining with primary antibody DAPI (DAKO, Carpentaria, CA) (Fig. 3).

Strong expression of endothelial cell specific proteins vWF and PECAM was confirmed by immunocytochemistry.

Measurement Example 3. Determination of Expression of Vascular Cell-Specific Protein by Fluorescence Active Flow Cytometry

Differentiation-induced ECs were separated into single cells by treatment with 0.05% trypsin-EDTA for 2 to 3 minutes for active flow cytometry. The single cells were washed with PBS containing 2% (v / v) fetal bovine serum (FBS; Gibco) for 20 minutes at 4 ° C. The cells were incubated with the antibody of phycoerythrin conjugated mouse anti-human stage-specific embryonic antigen-4 (SSEA-4), TEK tyrosine kinase-endothelial (Tie-2) and platelet endothelial cell adhesion molecule (BD Biosciences) Lt; / RTI > Isotype-matched IgG was used as a control and FACS Calibur (BD Biosciences) and CellQuest software (BD Biosciences) were used for data analysis.

In addition, protein expression of hESC-ECs was observed by the absence of SSEA-4, a pre-differentiation marker, and by the presence of EPC markers Tie-2 (11.29%) and PECAM (37.07%) ).

Measurement Example 4. Matrix analysis to determine vascularity of blood vessel cells

200 [mu] l of Matrigel (Corning, NY) were coagulated for 30 min at 37 [deg.] C in tissue culture plates. To induce angiogenesis, a suspension containing 200 μl baseline 1 × 10 ⁵ cells was placed on the coagulated matrigel and incubated at 37 ° C. in an overnight atmosphere. Immunocytochemistry using human PECAM, vWF and VE-cadherin was performed and the formation of the tube structure was observed through phase contrast microscopy (Fig. 5).

The in vitro function demonstrated by the angiogenesis assay identified in the differentiated endothelial cells is the capillary blood vessel which expresses PECAM, vWF and VE-cadherin and absorbs Ac-LDL (Acetylated low density lipoprotein; Biomedical Technologies, Stoughton, MA) Indicating the possibility of forming a similar structure.

Measurement example 5. Confirmation of vascular cell-specific expression of Ac-LDL

Absorption of AC-LDL was confirmed by incubation of differentiated hESC-ECs with 10 μg / ml DiI-labeled Ac-LDL for 4 h at 37 ° C. After incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes. Images were analyzed using a fluorescence microscope (Nikon) (Figure 6). As a control group, hCB-EPC (Human Cord Blood-derived Endothelial Progenitor Cell, cord blood-derived vascular precursor cells) was used.

Thus, the in vitro func- tionality was confirmed by the absorption of Ac-LDL.

Measurement Example 6. In vivo functional confirmation of differentiation-induced vascular cells

Measurement example 6-1. Visual analysis of symptoms

A Hindlimb ischemic mouse model was prepared according to a conventional method. Briefly, 6-week-old mice (Orientbio, Seoul, Korea) weighing 15 to 18 g were anesthetized and the skin was dissected to expose the femoral artery and separated from the femoral vein. Double knots were made in each of the femoral ligation sites on the side far from the center of the body through sutures. Then, a segment between the nodules was cut off and the incision site was sutured to induce ischemia in the mouse model. Bare limb ischemic mice after arterial surgery were randomly assigned to either of two experimental groups. Intramuscular transplantation was performed with a 29-tuber tuberculin syringe at four sites corresponding to the gracilis muscle located in the medial thigh for each group. The culture solution was injected into the saline group (n = 10) and 200 μl of EGM-2 MV ^™ (hESC-ECs group, n = 10) containing the hESC-ECs (3 × 10 ⁵ cells per mouse) ).

The function of the limb repair after inserting hESC-ECs into the lower limb ischemic mouse model was verified by comparing with the control group injected with saline. Both groups (n = 10) were observed for 4 weeks. In the transplanted hESC-ECs group, there was a significant reduction in the limb loss compared to the saline group. Saline loss (80%, 8/10) or severe limb necrosis (20%, 2/10) occurred in saline-infused mice and limb salvage Was not present. However, limb recovery (20%, 2/10) or mild limb necrosis (30%, 3/10) was observed in half of the hESC-EC transplanted group (Fig. 7).

Measurement example 6-2. Confirmation of treatment effect using Doppler laser measurement equipment

A laser Doppler vascular perfusion imaging device (Moor Instruments, Devon, UK) was used for sequential, noninvasive physiological demonstration of angiogenesis. The treated mice were scanned for blood flow on the hindlimb surface sequentially at 0, 7, 14, and 28 days, respectively. Quantitative analysis of blood flow was performed at the knee joint to foot region using images displayed in digital color, and the perfusion average value was calculated (FIG. 8). For the measurement, the Doppler device analysis software was used. The measurement values for each group were expressed as a ratio between 0 and 1 based on the normal group.

During the 28th day, limb loss or severe necrosis occurred in the control group, whereas half of the transplantation group had the effect of restoring blood flow from ischemic symptoms.

After one week of treatment, the relative blood flow rate was 0.272 ± 0.017 in the hESC-EC transplant group and 0.133 ± 0.003 in the saline infusion group. After 2 weeks, 0.471 ± 0.045 in the hESC-EC transplant group and 0.136 ± 0.009 in the saline infusion group. After 3 weeks there was 0.551 ± 0.067 in the hESC-EC transplant group and 0.14 ± 0.019 in the saline infusion group. After 4 weeks, the hESC-EC transplantation group was 0.588 ± 0.014 and the saline infusion group was 0.145 ± 0.003.

Measurement example 6-3. Confirmation of treatment effect using microcomputer

The blood vessels of the rat was fixed to ^{Microfil ® (MV-120, Flow} Tech, Inc., Carver, MA) in order to take advantage of the blood perfusion system (Thermo Fisher Scientific, Waltham, MA ). The transplantation site was fixed with 4% formalin for 28 days and radiographic analysis was performed on a computerized tomography scanner (Skyscan1172, Bruker-microct, Kontich, Belgium). The CT images of the scanned samples were taken in 3D (three-dimensionally) constructed from computer software (NRecon; Skyscan, Aartselaar, Belgium) at a resolution of 50 mm (100 kV and 100 mA). The extent of lesions in the regenerated microvessels, muscle mass, and injury sites were calculated from images constructed in 3D using CTAn software (Skyscan).

Computed tomography was performed to visualize the vascular structure and angiogenesis pattern evidenced during vascular reconstruction and to confirm the regeneration process from graft ischemia to normal conditions in the graft group. The red arrows indicate arterioles and capillaries, and the yellow arrows indicate vessels (FIG. 9).

Measurement example 6-4. Identification of treatment effect using tissue immunochemistry analysis

To perform histological analysis of muscle reconstruction and fibrosis, ischemic limb tissue was resected from mice after 4 weeks. Samples were fixed with buffer containing 10% (v / v) formaldehyde, dehydrated with graded ethanol series (graded ethanol series) and added to paraffin. The dehydration process was performed sequentially for 1 hour using 70, 80, 90, 100 and 100% ethanol. Samples were sliced with 4 μm thick sections and stained with hematoxylin and eosin (H & E). Collagen staining using Masson's trichrome (MT) was also performed to confirm the presence of fibrosis in ischemic tissues (FIG. 10A). To determine the presence of human cells, tissue sections were stained with immunohistochemistry using anti-human smooth muscle actin (SMA; BD Bioscience) and anti-human PECAM antibodies (BD Bioscience) 10B). Staining was visualized using the ^{avidin-biotin complex immunoperoxidase (Vectastain ®} ABC kit), 3,3'-diaminobenzidine (DAPI), substrate solution kits (Vector Laboratories, Burlingame, CA).

H & E staining was used to visualize tissue and measure the extent of muscle regeneration. The hESC-ECs transplantation group showed a significant muscle reconstruction effect, indicating that the regeneration effect was improved compared to the control group.

MT staining revealed that hESC-ECs transplantation group enables tissue regeneration as the most active collagen accumulation occurs. Conversely, in the control sample injected with saline, fibrosis was severe and there was no possibility of muscle regeneration.

In order to quantitate the volume (mm ² ) of the small arteries and capillaries, SMA and PECAM staining showed significant vessel volumes in the hESC-EC graft group. Quantitative analysis revealed that neovascularization was significantly enhanced compared with the control group. The volumes of the small arteries and capillaries were 17 ± 4 mm ² and 293.3 ± 45.1 mm ² in the saline group and 37 ± 3 mm ² and 696.6 ± 49.3 mm ² in the transplant group respectively.

Measurement example 6-5. Tracking human vascular cells in animals

To perform fluorescence in situ hybridization (FISH), the sample slices were deparaffinized by treatment with xylene at room temperature, dehydrated using 100% ethanol, and dried. The paraffin pretreatment solution was pre-heated to 95 ° C in a heating mantle, soaked for 30 minutes, and then immersed in 2X standard saline citrate (SSC) twice for 5 minutes each. 500 μl of protease was added to 50 ml of protease buffer preheated to 37 ° C to prepare a protease solution, and the sample slice was immersed for 20 minutes. Then, the sections were immersed again in 2X SSC, dehydrated in 70% ethanol for 1 minute, and again in 100% ethanol for 1 minute. After drying in air, a hybridizing area was marked with a diamond pen. CEP 17 DNA probe (2 μl, Vysis, Downers Grove, IL) was applied before the cover glass was sealed with a rubber adhesive. As a result, a hybridization was carried out in a humid space at 37 ° C under an over night condition using a probe protected from light and denatured at 75 ° C for 5 minutes. After hybridization, the rubber adhesive was removed and the slice samples were washed in 4X SSC containing 0.1% NP (nonyl phenoxypolyethoxylethanol, NP-40) for 10 min at 42 ° C in 50% (v / v) formaldehyde / 2X SSC I soaked for 5 minutes. The slice was dried in air without light, and stained with DAPI (Fig. 11).

FISH analysis confirmed that the transplanted EC cells carried a human genome (white arrow).

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Treating the stem cells with Dulbecco's phosphate buffered saline (DPBS) to form a single cell; And a step of suspension culture of the single cells to form an aggregate.

The method according to claim 1,
Further comprising the step of dissociating the aggregate and causing adhesion culture.

3. The method of claim 2,
Wherein the adherent culture is performed in a collagen medium.

The method according to claim 1,
The stem cells may be selected from the group consisting of embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), somatic cell nuclear transfer (SCNT), and pluripotent stem cells PSC). &Lt; / RTI > The method of claim 1, wherein the stem cell-derived vascular cell non-purified differentiation is selected from the group consisting of:

(1) single cellization of stem cells by treating with DPBS;
(2) suspending the single cells to form aggregates;
(3) classifying the aggregate of step (2) into an angiogenic differentiation group and a hemangioblast differentiation group; And
(4) A method for co-differentiation of stem cell-derived vascular cells and hematopoietic stem cells, comprising the step of differentiating two groups of aggregates classified by the above step (3).

6. The method of claim 5,
Wherein the step of differentiating the agglutinating group of the vascular cell differentiation group in the step (4) is a step of dissociating and co-culturing the agglutinin and the vascular cell differentiation group.

The method according to claim 6,
Wherein the adherent culture is performed in a collagen culture medium.

6. The method of claim 5,
Wherein the step of differentiating the aggregates of the differentiating group of hematopoietic cells in step (4) is a step of suspending the aggregates, and the step of differentiating the aggregates of the hemopoietic stem cell-derived vascular cells and the hematopoietic stem cells.

6. The method of claim 5,
Wherein the stem cell is any one selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, somatic cell cloning stem cells, and pre-differentiating stem cells, and the method for simultaneous differentiation of stem cell-derived vascular cells and hematopoietic stem cells.