WO2017122887A1 - Method for simultaneous differentiation of stem cell-derived endothelial cells and hemangioblasts without purification - Google Patents

Abstract

The present invention relates to a method for the simultaneous differentiation of stem cell-derived endothelial cells and hemangioblasts without purification, the method comprising the steps of: treating stem cells with DPBS to form single cells; suspension culturing the single cells to form aggregates; classifying the aggregates into an endothelial cell differentiation group and a hemangioblast differentiation group; and differentiating the aggregates in the two classified groups. In addition, unlike a conventional method which requires a purification process for selecting desired cells in a cell differentiation process, the present invention does not require a separate purification process, thereby reducing cost and time.

Description

Simultaneous Differentiation of Stem Cell-Derived Vascular and Hematopoietic Cells

The present invention relates to a method for simultaneously differentiating stem cell-derived vascular cells and hematopoietic stem cells, and more specifically, by using DPBS, a population of embryonic stem cells can be dissociated into single cells, followed by suspension culture to separate vascular cells and hematopoietic cells, respectively. A method for differentiation without purification.

Stem Cells are cells that have the ability to differentiate into various types of cells, and are used to grow into alternative tissues, which have the ability to grow on their own as one of the many cell types present in the body. It is used and studied in the treatment of various diseases.

Among them, human embryonic stem cells are cells derived from the inner cell mass of human blastocysts and have autologous proliferation and differentiation potential. Induction of differentiation into specific cells using human embryonic stem cells has been used to induce differentiation into specific cells or to process specific cytokines through the Embryoid body stage, which is characterized by triodenal characteristics. .

The development of endothelial cells is differentiated from hemangioblasts and angioblasts, which are mesodermal cells of trioderm, and form blood vessels through vasculogenesis and angiogenesis.

The process of making blood vessels in the human body is largely divided into two types: angiogenesis mechanisms formed by cells in which vascular endothelial cells move and grow from surrounding blood vessels, and angiogenesis mechanisms in which blood cells circulate in tissues to form blood vessels. Are separated by paths. In 1997, Asahara et al. At the Elizabeth Medical Center at Tufts University discovered that blood vessels were formed by vascular endothelial progenitor cells.

Vascular cells play an important role in angiogenesis and are involved in the maintenance and maintenance of all tissues in vivo, angiogenesis, inflammation and thrombosis. In order to prepare such vascular cells, human embryonic stem cells in undifferentiated state are induced into embryos, and then an antibody that expresses vascular cell-specific antibodies is immunized and separated by laser (FACS) and magnet (MACS) methods. Although it has been written, it has been limited in that the method is difficult and time-consuming and expensive.

Related well-known technologies include Korean Patent Registration No. 10-0948170 (2010.03.10.) And Korean Patent Application Publication No. 10-2013-0105124 (2013.09.25.).

The present inventors devised a cell differentiation method by mimicking the process of cell differentiation in human body, and completed the present invention by simultaneously obtaining hematopoietic and vascular cells according to the cell differentiation method using DPBS.

An object of the present invention, by differentiating hematopoietic cells and vascular cells simultaneously using a method designed to mimic the process of differentiation of human cells in order to more easily perform differentiation of hematopoietic cells and vascular cells, saving time and money To provide a way to do it.

In order to achieve the above object, the present invention comprises the steps of treating a single cell to DPBS stem cells; And it provides a stem cell-derived vascular cell non-purified differentiation comprising the step of floating culture the single cells to form aggregates.

In another aspect, the present invention provides a method for stem cell-derived vascular cell undifferentiated differentiation, further comprising the step of dissociating the aggregates to adhere to the culture.

In one embodiment of the present invention, the adhesion culture may be carried out in collagen medium.

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

In addition, the present invention (1) the step of treating single cells by treating DPBS to stem cells; (2) floating the single cells to form aggregates; (3) classifying the aggregate of step (2) into a vascular cell differentiation group and a hematopoietic differentiation group; And (4) provides a method for simultaneously differentiating stem cell-derived vascular cells and hematopoietic stem cells comprising the step of differentiating the aggregates of the two groups classified by step (3).

In one embodiment of the present invention, the step of differentiating the aggregates of the vascular cell differentiation group in the step (4) may be a step of dissociating the aggregates to adhere culture.

In one embodiment of the present invention, the adhesion culture may be carried out in collagen medium.

In one embodiment of the present invention, the step of differentiating the aggregates of the hematopoietic differentiation group in the step (4) may be a step of floating culture the aggregates.

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

According to the present invention as described above, by using single-celled embryos to form agglomerates and then aggregated embryos using DPBS on the stem cells, the aggregates are single-celled to differentiate vascular cells or resuspended culture blood Since differentiation of blast cells, there is an effect that can simultaneously differentiate blood vessels and hematopoietic cells.

In addition, according to the present invention, unlike the conventional cell differentiation process, which requires a purification process to select the desired cells, it does not require a separate purification process, thereby reducing the cost and time.

1 is (A) dated progression, (B) embryonic body formation process from stem cells, (C) vascular cell formation process using embryonic bodies for the differentiation process of vascular cells and hematopoietic cells according to an embodiment of the present invention And (D) Schematic diagram showing the process of blood stem cell formation using the embryoid body.

Figure 2 is a photograph showing the characteristics of vascular cells differentiated according to an embodiment of the present invention through the RT-PCR as the expression level of the gene.

Figure 3 is a photograph showing the expression of differentiated vascular cell specific proteins vWF and PECAM according to an embodiment of the present invention through immunocytochemistry.

Figure 4 is a photograph showing the expression levels of vascular cell specific proteins differentiated according to an embodiment of the present invention (A) SSEA-4, (B) Tie-2, and (C) PECAM through fluorescence activated flow cytometry.

Figure 5 is a photograph confirming the ability to form the tube structure by confirming the expression of vascular cell specific proteins (A) PECAM, (B) vWF and (C) VE-cadherin differentiated according to an embodiment of the present invention.

Figure 6 is a photograph showing a comparison of the expression level of vascular cell-specific Ac-LDL differentiated according to an embodiment of the present invention in hESC-EC and hCB-EPC.

Figure 7 is a photograph and table comparing the symptoms of the ischemic mouse model infused with differentiated vascular cells according to an embodiment of the present invention.

Figure 8 is a (A) picture and (B) graph showing the therapeutic effect of the lower limb ischemic mouse model injected with differentiated vascular cells in accordance with an embodiment of the present invention using the Doppler laser equipment.

9 is a micro-CT image showing the therapeutic effect of the ischemic mouse model infused with differentiated vascular cells according to an embodiment of the present invention.

10 is a (A) picture and (B) graph of the therapeutic effect of the model of the ischemia of the lower limb ischemia injected with differentiated vascular cells according to an embodiment of the present invention.

Figure 11 is a photograph showing the therapeutic effect of the lower limb ischemic mouse model injected with differentiated vascular cells in accordance with an embodiment of the present invention through a fluorescent in situ test.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of treating single stem cells by treating Dulbecco's phosphate buffered saline (DPBS) on stem cells; It provides a stem cell-derived vascular cell (Endothelial Cell) non-purified differentiation method comprising the step of suspension culture (suspension culture) to form an aggregate.

Suspension culture is a culture method performed in a state in which cells are suspended in a culture medium during the culture of animal cells. Cells that proliferate in a floating state in vivo, such as blood cells or a plurality of cancer cells, can be suspended in culture without shaking or rotating, but in most cases, they are rotated (eg, incubated) or shaken in each culture bottle. Or it may be necessary to rotate the incubator.

In another aspect, the present invention provides a method for stem cell-derived vascular cell differentiation, further comprising the step of dissociating the aggregates and adhering culture.

Adhesion culture is a method applied to general animal cells and is a culture method that induces growth in a monolayer while cells are attached on a solid (agar) medium. This is also known as monolayer culture.

In one embodiment of the present invention, the adhesion culture may be performed in collagen (Collagen) medium.

In one embodiment, the stem cells are embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), somatic cell nuclear transfer (SCNT) and It may be any one selected from the group comprising pluripotent stem cells (PSC).

In addition, the present invention (1) the step of treating single cells by treating DPBS to stem cells; (2) floating the single cells to form aggregates; (3) classifying the aggregate of step (2) into a vascular cell differentiation group and a hemangioblast differentiation group; And (4) provides a method for simultaneously differentiating stem cell-derived vascular cells and hematopoietic stem cells comprising the step of differentiating the aggregates of the two groups classified by step (3).

When enzymatic treatment is used for single dissociation of cells, many cells suffer from cell death or damage. In the case of treatment with DPBS, it was confirmed that the cell death rate can be improved since cell death is not induced and ultimately, the differentiation of cells can be efficiently proceeded and thus applied.

Preferably the DPBS according to the present invention can be used that does not contain calcium and magnesium. The use of DPBS, which contains calcium and magnesium for cell dissociation, reduces cell dissociation.

In one embodiment of the present invention, the step of differentiating the aggregates of the vascular cell differentiation group in the step (4) may be a step of dissociating the aggregates to adhere culture.

In one embodiment of the present invention, the adhesion culture may be carried out in collagen medium.

In one embodiment of the present invention, the step of differentiating the aggregates of the hematopoietic differentiation group in the step (4) may be a step of floating culture the aggregates.

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

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Culture of Human Embryonic Stem Cells

The culture medium for culturing human embryonic stem cells consisted of 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 lines. Human embryonic stem cells were subcultured every 5 to 7 days by co-culture of mouse embryonic fibroblasts (MEF) inactivated cell division as feeder cells (Co-culture).

Example 2. Aggregate Formation Using Stem Cells

The cell differentiation process of the present invention suggests a method of dissociating into single cells after embryonic formation and then forming an aggregate to simultaneously differentiate into two cells (FIG. 1A).

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

Looking at the method in more detail, the cell culture solution was incubated (suction), the cells were washed once with DPBS, 5 ml of DPBS was added and incubated for 5 to 10 minutes in a CO ₂ atmosphere. The phenomenon of dissociation of stem cell colonies under a microscope was confirmed, and pipetting was performed lightly so as not to damage cells.

Then, when human embryonic stem cell colonies were dissociated into single cells, they were placed in a 40 um strainer and separated into pure single cells. This was centrifuged at a speed of 1000 ~ 1500 rpm to remove the supernatant.

Stemline II culture solution (Sigma-Aldrich) and VEGF (Vascular endothelial growth factor, vascular epidermal growth factor; Peprotech) 50 ng / ml, BPP4 50 ng / ml were added to the isolated single cells. Aggregates were formed by suspension culture for 2 days at.

After 2 days, 50% of the culture solution was removed, and then 50% of the Stemline II culture medium and VEGF 50 ng / ml, BMP4 50 ng / ml, TPO 50 ng / ml, Flt-3 50 ng / ml, SCF 50 ng The culture was replaced by adding / ml, 50 ng of bFGF. After incubation for 2 more 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 (Collagen type IV) was coated on a 60 mm plate, and the dissociated single cell 5 x 10 ⁵ cells were added. According to this method, when the cell density in the collagen-coated plate became 80 to 90% or more, it took 2 to 3 days to generate human embryonic stem cell-derived vascular cells by subculture (FIG. 1C).

Example 4 Differentiation of Hematopoietic Cells Using Aggregates

Methylcellulose H4436 (methylcellulose) comprising additives such as BMP4, SCF, VEGF, TPO, Flt-3, bFGF and EX-CYTE (Millipore, Billerica, Mass.) To characterize the aggregate prepared in Example 2 Stemcell Technologies, Vancouver, British columbia, Canada) was added to each well of a 6 well dish at a concentration of 5 x 10 ⁵ cells. After 2-3 days, the grape cluster shape, which is characteristic of hematopoietic cells, appeared, and after 6-7 days, differentiation was completed (FIG. 1D).

Measurement Example 1. Analysis of Expression of Vascular Cell Specific Marker Genes by RT-PCR

To analyze the expression level of RNA, total RNA was extracted using a NanoDrop ^® ND-1000 spectro photometer (NanoDrop Technologies, Wilmington, DE) using Trizol ^® (Invitrogen, Carlsbad, Calif.). First, reverse transcription was performed using Maxim RT pre mix kit (iNtRon biotechnology, Sungnam, Korea) to obtain cDNA. Analysis via 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 of 30 seconds at 95 ℃, 30 seconds at 55 ~ 60 ℃, 30 seconds at 72 ℃. The PCR cycle began with a 5 minute initial denaturation step at 95 ° C. and ended with a 10 minute final expansion step at 72 ° C. RT-PCR primer sequences are listed in Table 1. RT-PCR results were confirmed by 1.5% agarose gel (Promega, Madison, Wis.) By staining with EtBr (ethidium bromide) (Fig. 2).

Table 1

RT-PCR was performed to determine whether differentiation was induced through decreased expression of genes (oct-4 and nanog) exhibiting pluripotency and thus increased expression of endothelial markers. Pluripotent genes expressed in hESCs were strongly expressed until day 2 hEBs, but were not detected in hESC-derived ECs with further differentiation. Similar to the control CB-EPC, induced ECs resulted in the endothelial progenitor cell (EPC) markers flt-1, tie-2, VE-cad, vwf and kdr (kinase insert domain receptor) on day 7 ) Is expressed.

Measurement Example 2 Confirmation of Expression of Vascular Cell Specific Proteins through Immunocytochemical Analysis

The 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 30 minutes of 5% normal goat serum, the cells were treated with primary antibodies such as anti-human platelet endothelial cell adhesion molecule (PECAM) and anti-human van Willebrand factor (vWF) at 4 ° C. 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 cell nuclei were visualized by staining with a primary antibody, DAPI (DAKO, Carpentaria, CA) (FIG. 3).

Strong expression of endothelial cell specific proteins, vWF and PECAM, was confirmed immunohistochemically.

Measurement Example 3 Confirmation of Expression of Vascular Cell- Specific Proteins by Fluorescent Active Flow Cytometry

Differentiation-induced ECs were treated with 0.05% trypsin-EDTA for 2-3 minutes for active flow cytometry and isolated into single cells. The single cells were washed at 4 ° C. for 20 minutes with PBS containing 2% (v / v) fetal bovine serum (FBS; Gibco). The cells were then treated with antibodies of phycoerythrin (PE) -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). Incubated together. Isotype-matched 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 active flow cytometry indicating the absence of the pluripotency marker SSEA-4 and the presence of the EPC markers Tie-2 (11.29%) and PECAM (37.07%) (FIG. 4). ).

Measurement Example 4 Confirmation of Angiogenesis of Vascular Cells by Matrigel Analysis

200 μl of Matrigel (Corning, NY) was coagulated for 30 minutes at 37 ° C. in tissue culture plates. A suspension containing 200 μl 1 × 10 ⁵ cells to induce angiogenesis was placed on the coagulated Matrigel and incubated at 37 ° C. in overnight conditions. Immunocytochemistry using human PECAM, vWF and VE-cadherin revealed tube structure formation through phase contrast microscopy (FIG. 5).

In vitro functions demonstrated through angiogenesis assays identified in differentiated endothelial cells are capillaries that express PECAM, vWF and VE-cadherin and absorb Ac-LDL (Acetylated low density lipoprotein; Biomedical Technologies, Stoughton, Mass.) Indicates the likelihood of similar structure formation.

Measurement Example 5 Confirmation of Expression of Vascular Cell-Specific Ac-LDL

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

Accordingly, the in vitro functional status was confirmed through the absorption of Ac-LDL.

Measurement Example 6. In Vivo Functionality of Differentiated Induced Vascular Cells

Measurement Example 6-1. Visual analysis of symptoms

Hindlimb ischemic mouse models were constructed according to conventional methods. Briefly, 6 week old mice (Orientbio, Seoul, Korea) weighing 15-18 g were anesthetized and separated from the femoral vein after incision of the skin to expose the femoral artery. Sutures made double knots in each of the femoral ligation sites far and near the center of the body. Then, a segment between the nodules was cut and the incision site was closed to induce leg ischemia in the mouse model. The ischemic mice after arterial surgery were randomly assigned to either of two experimental groups. Intramuscular transplantation was performed using a 29-caliber tuberculin syringe at four sites corresponding to the gracilis muscle located in the medial thigh for each group. The saline group (n = 10) was injected with culture and the experimental group received 200 μl of EGM-2 MV ^TM (hESC-ECs group, n = 10) containing the previously obtained hESC-ECs (3 × 10 ⁵ cells per mouse). ) Was injected.

In vivo function was confirmed by comparing the effect of limb repair after insertion of hESC-ECs on the ischemic mouse model with the control group injected with saline. Observations were made for 4 weeks for both groups (n = 10). The intramuscular transplantation of hESC-ECs showed a significant reduction in limb loss compared to the saline group. Mice in the saline-infused group showed limb loss (80%, 8 out of 10) or severe limb necrosis (20%, 2 out of 10) and limb salvage Did not exist. However, in half of the groups implanted with hESC-EC, limb recovery (20%, 2 out of 10) or mild limb necrosis (30%, 3 out of 10) was observed (FIG. 7).

Measurement Example 6-2. Confirmation of therapeutic effect using Doppler laser measurement equipment

Laser Doppler Vascular Perfusion Imaging (Moor Instruments, Devon, UK) was used for sequential and noninvasive physiological demonstration of angiogenesis. Mice after the condition treatment were observed by sequentially scanning the blood flow on the hind limb surface every 0, 7, 14 and 28 days. Quantitative analysis of blood flow at the site of the knee joint to the foot was performed using the digitally displayed image and the perfusion average value was calculated (FIG. 8). The analysis software of the Doppler device was used for the measurement, and the measurement value of each group was expressed as a ratio between 0 and 1 based on the normal group.

For 28 days, there was an effect of limb loss or severe necrosis in the control group, whereas half of the transplant group recovered blood flow from the symptoms of lower limb ischemia.

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, it was 0.471 ± 0.045 in the hESC-EC transplant group and 0.136 ± 0.009 in the saline infusion group. After 3 weeks, it was 0.551 ± 0.067 in the hESC-EC transplant group and 0.14 ± 0.019 in the saline infusion group. After 4 weeks, it was 0.588 ± 0.014 in the hESC-EC transplant group and 0.145 ± 0.003 in the saline infusion group.

Measurement Example 6-3. Confirmation of treatment effect using micro computed tomography

The blood vessels of mice were fixed with Microfil ^® (MV-120, Flow Tech, Inc., Carver, MA) to use a blood perfusion system (Thermo Fisher Scientific, Waltham, Mass.). The implantation site was fixed with 4% formalin for 28 days and radiographic analysis was performed with a computed tomography device (micro-CT scanner, Skyscan1172, Bruker-microct, Kontich, Belgium). CT images of the scanned samples were taken in three-dimensionally (3D) built from computer software (NRecon; Skyscan, Aartselaar, Belgium) at a resolution of 50 mm (100 kV and 100 mA). The reconstructed microvascular and muscle volume and the extent of injury were calculated from 3D images using CTAn software (Skyscan).

Computed tomography was performed to visualize vascular structure and angiogenesis patterns during vascular reconstruction, to identify the regeneration process approaching normal conditions from lower limb ischemia in the transplant group. Red arrows mark arterioles and capillaries and yellow arrows mark blood vessels (FIG. 9).

Measurement Example 6-4. Confirmation of treatment effect using tissue immunochemical analysis

To perform histological analysis for muscle reconstruction and fibrosis, ischemic limb tissue was excised from mice after 4 weeks. Samples were fixed with buffer containing 10% (v / v) formaldehyde, dehydrated with progressively high concentrations of ethanol (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 into 4 μm thick sections and stained with hematoxylin and eosin (H & E). Collagen staining using Masson's trichrome (MT) was also performed to confirm fibrosis in ischemic tissues (FIG. 10A). To confirm the presence of human cells, tissue sections were stained by immunohistochemistry using anti-human smooth muscle actin (SMA; BD Bioscience) and anti-human PECAM antibodies (BD Bioscience) (FIG. 10B). Staining results were visualized using avidin-biotin complex immunoperoxidase (Vectastain ^® ABC kit), 3,3'-diaminobenzidine (DAPI), and 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 transplant group showed significant muscle reconstruction effects, indicating that the regenerative effect was improved compared to the control.

MT staining revealed that collagen accumulation was most active in the hESC-ECs transplant group, enabling tissue regeneration. In contrast, saline-injected control samples showed severe fibrosis and no muscle regeneration potential.

In order to quantitatively measure the volume of the arterioles and capillaries (mm ² ), SMA and PECAM staining showed significantly higher blood vessel volume in the hESC-EC transplant group. Quantitative analysis confirmed that neovascularization was significantly enhanced compared to the control group. The arterial and capillary volumes 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 blood vessels transplanted into animals

In order to perform Fluorescence in situ hybridization (FISH), sample sections were treated with xylene at room temperature, deparaffinized, dehydrated using 100% ethanol, and dried. The paraffin pretreatment solution was preheated to 95 ° C. in a heating mantle and soaked for 30 minutes, and then twice each for 5 minutes in 2X standard saline citrate (SSC). Protease solution was prepared by adding 500 μl of protease to 50 ml of protease buffer preheated to 37 ° C. and soaking the sample sections for 20 minutes. Then, the sections were again immersed in 2X SSC, soaked in 70% ethanol for 1 minute, and dehydrated in 100% ethanol again for 1 minute. After drying in air, the hybridizing area was marked using a diamond pen, and CEP 17 DNA probe (2 μl, Vysis, Downers Grove, IL) was applied before sealing the cover glass with rubber adhesive. As a result, hybridization was performed in a humid space at 37 ° C. under overnight conditions using a probe protected from light and denatured at 75 ° C. for 5 minutes. After the hybridization process, the rubber adhesive was removed and the section samples were washed at 50 ° C. with 50% (v / v) formaldehyde / 2X SSC for 10 minutes at 42 ° C. to 4X SSC containing 0.1% nonyl phenoxypolyethoxylethanol (NP-40). Soak for 5 minutes. Sections were dried in the air without light and contrast-stained using DAPI (FIG. 11).

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

As mentioned above, specific portions of the present disclosure have been described in detail, and it is apparent to those skilled in the art that such specific techniques are merely preferred embodiments, and thus the scope of the present disclosure is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

1. Treating single stem cells by treating Dulbecco's phosphate buffered saline (DPBS) on stem cells; And stem cell-derived vascular cells (Endothelial Cell) differentiation method comprising the step of suspending culture to form agglomerates.
2. The method of claim 1,

   Stem cell-derived vascular cell non-differentiated differentiation method further comprising the step of dissociating the aggregate to an adhesion culture.
3. The method of claim 2,

   Stem cell-derived vascular cell undifferentiated differentiation method, characterized in that the adhesion culture is performed in collagen (Collagen) medium.
4. The method of claim 1,

   The stem cells are embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), somatic cell nuclear transfer (SCNT) and pluripotent stem cells, Stem cell-derived vascular cell non-purified differentiation method, characterized in that any one selected from the group containing.
5. (1) treating the stem cells with DPBS to single cell;

   (2) floating the single cells to form aggregates;

   (3) classifying the aggregate of step (2) into a vascular cell differentiation group and a hemangioblast differentiation group; And

   (4) A method for simultaneously differentiating stem cell-derived vascular cells and hematopoietic stem cells, comprising differentiating aggregates of two groups classified by step (3).
6. The method of claim 5,

   In step (4), the step of differentiating aggregates of the vascular cell differentiation group is a step of dissociating the aggregates and attaching and culturing stem cell-derived vascular cells and hematopoietic cells.
7. The method of claim 6,

   Simultaneous differentiation of stem cell-derived vascular cells and hematopoietic stem cells, characterized in that the adhesion culture is performed in collagen medium.
8. The method of claim 5,

   In step (4), the step of differentiating the aggregates of the hematopoietic differentiation group is a step of floating culture of the aggregates, characterized in that the stem cell-derived vascular cells and hematopoietic cells undifferentiated simultaneous differentiation method.
9. The method of claim 5,

   Simultaneous differentiation method of the stem cell-derived vascular cells and hematopoietic stem cells, characterized in that the stem cells are any one selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, somatic clonal stem cells and pluripotent stem cells.

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