KR101045492B1 - Therapeutic implant for wound-healing using human embryonic stem cell derived vascular angiogenic progenitor cellshESC-VAPCs and method thereof - Google Patents

Abstract

The present invention (a) culturing the vascular progenitor cells derived from embryonic stem cells for transplantation; (b) providing a wound treatment method using the embryonic stem cell-derived vascular progenitor cells, comprising transplanting the cultured embryonic stem cell-derived vascular progenitor cells into the skin of a mammal.

The wound treatment method of the present invention eliminates limitations on cell sources by using human embryonic stem cell-derived vascular precursor cells, which are new cell sources of vascular precursor cells known to be effective in treating existing wounds. It is effective to greatly improve the wound treatment efficiency than conventional adult stem cell-derived vascular precursor cells. In addition, according to the wound treatment method of the present invention, transplanted human embryonic stem cell-derived vascular progenitor cells promote angiogenesis to facilitate the transfer of factors necessary for wound healing, thereby increasing the synthesis of collagen required for wound healing. The newly formed collagen layer creates an environment in which stromal cells, skin cells, and the like, which are necessary for wound healing, can be more easily grown, thereby shortening the wound healing period.

Human embryonic stem cells, wound treatment, vascular precursor cells derived from human embryonic stem cells

Description

Therapeutic implant for wound-healing using human embryonic stem cell derived vascular angiogenic progenitor cells (hESC-VAPCs) and method

The present invention relates to a wound treatment technique using embryonic stem cell-derived vascular progenitor cells, and more particularly, to a wound treatment technique using human embryonic stem cell-derived vascular progenitor cells, derived from human embryonic stem cells cultured at high concentration in the wound site. By implanting vascular progenitor cells, vascular progenitor cells promote angiogenesis, thereby effectively treating wound sites in a shorter time than conventional adult stem cell derived vascular progenitor cells.

Currently wound healing is widely practiced through artificial skin transplantation.

Artificial skin, which is applied to wounds with skin defects during large burns or surgical procedures, consists of synthetic polymers and natural polymers.Since the upper layer of silicone prevents the body fluid from evaporating and disappearing, the lower layer is composed of collagen or sulfate chondroitin. Induces the regeneration of blood vessels and connective tissues, and by using the patient's own cells, the cells constituting each layer of the skin are collected and expanded in vitro, and then inoculated into artificial skin made of appropriate synthetic or natural polymers. Induction of formation and transplantation back to the patient has been used.

However, the treatments using artificial skin have not been able to produce a certain result in terms of wound recovery. Especially in the case of cultured skin, epidermal cells are difficult to culture well enough to stabilize patient tissues. Not only do epidermal cells grow poorly on human-derived fibroblasts, but also have fatal drawbacks in that the epidermal and dermal bonds are unstable during engraftment.

Normal wound healing takes place in several stages, but all processes of coagulation, inflammation, cell cycle and tissue reorganization after wounding are ultimately driven by blood circulation in the local wound area. This local blood circulation is promoted by the formation of new blood vessels and carries various growth factors, inflammatory cells, stromal cells, and skin precursor cells necessary for wound recovery through the newly formed blood vessels, and also removes tissue debris and forms granulation tissue. It is known to promote.

In this respect, the effect of wound healing by transplantation of vascular progenitor cells with neovascularization ability can be expected, and the effects of wound treatment using vascular precursor cells derived from adult stem cells have been reported.

Kim WS and others have demonstrated the effects of wound healing using adipose stem cell-derived cells (Won-Serk Kim, Byung-Soon Park, Jong-Hyuk Sung, Jun-Mo Yang, Seok-Beom Park, Sahng-June Kwak). , Jeong-Soo Park, Wound healing effect of adipose-derived stemcells: A critical role of secretory factors on human dermal fibroblasts (2007) Journal of Dermatological Science 48, 15-24), Stephen M et al. The cells were used to demonstrate their effectiveness in treating wounds caused by ischemic diseases (Stephen M. Bauer, Lee J. Goldstein, Richard J. Bauer, Haiying Chen, Mary Putt, ScD, and Omaida C. Velazquez, The bone marrow- derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia.Journal of vascular surgery (2006) 43 (1): 134-41), Suh W et al. have demonstrated the effectiveness of wound healing using cord blood stem cell-derived vascular precursor cells. Suh W, Kim KL, Kim JM, Shin IS, LEE YS, Jang HS, LEE JS, Byun J, Choi JH, Jeon ES, Kim DK, Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes / macrophages and neovascularization (2005) 23 (10): 1571-8).

However, yields of vascular progenitor cells from adult stem cells such as normal bone marrow or umbilical cord blood are extremely low, and an alternative is needed. Therefore, the use of human embryonic stem cells with infinite proliferation and differentiation potential could be developed as an alternative to new cell therapies.

Human embryonic stem cells are cells with infinite self-renewality and pluripotency capable of differentiating into trigeminal cells (endoderm, mesoderm, ectoderm) that make up the human body within a certain environment ( Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM, Embryonic stem cell lines derived from human blastocysts.Science (1998) 282: 1145-1147), vascular cells, nerve cells, muscle As studies inducing differentiation into cells, pancreatic cells, etc. have been published, cells with great potential for the treatment of intractable diseases have been recognized as important cells in various fields.

Therefore, in order to develop a cell therapeutic agent for wound treatment, there is an urgent need to provide more innovative and fundamental therapeutic methods through application to wound animal models using human embryonic stem cell-derived vascular precursor cells.

The present inventors conducted various studies for the development of wound treatment technology using human embryonic stem cell-derived vascular progenitor cells.

As a result, when the human embryonic stem cell-derived vascular progenitor cells are proliferated / cultured to the extent possible to treat the disease model and transplanted into the wound disease animal model, wounds are more effectively performed in a shorter time than the umbilical cord blood-derived vascular progenitor cells which are currently widely used. A decrease in site size was found. In addition, as a result of histological analysis in the human embryonic stem cell-derived vascular stem cell treatment group, it was found that a larger amount of collagen layer was formed than in the culture medium treatment group, and re-epithelialization was promoted faster.

Accordingly, an object of the present invention is to provide a more effective wound treatment technology using human embryonic stem cell-derived vascular progenitor cells.

The present invention (a) culturing the vascular progenitor cells derived from embryonic stem cells for transplantation; (b) providing a wound treatment method using the embryonic stem cell-derived vascular progenitor cells, comprising transplanting the cultured embryonic stem cell-derived vascular progenitor cells into the skin of a mammal.

The present invention uses human embryonic stem cells as embryonic stem cells, and provides a wound treatment method using vascular progenitor cells derived therefrom.

Human embryonic stem cells used for the culture are differentiated through the formation of embryoid bodies in a hypoxic state to separate vascular progenitor cells, and then concentrated embryonic stem cell-derived vascular precursor cells by flow cytometry (FACS) using a marker. The human embryonic stem cells to be used are preferable, and the marker used for flow cytometry is preferably a double marker of CD133 / KDR.

In the step (a), in order to effectively proliferate and culture the human embryonic stem cell-derived vascular progenitor cells, the culture dish is preferably coated with a collagen and then cultured, and the culture medium is cultured using an EGM-2 MV culture solution. Preferably, if necessary, known cultures such as trypsin-EDTA, DMEM culture, M199 culture, or a mixed culture thereof may be used, and cell proliferation may be propagated by subculture, and each of the necessary cultures may be selected for each subculture. Can be cultured.

In step (b), the embryonic stem cell-derived vascular progenitor cells are preferably transplanted with matrigel to form a support for the cells to be transplanted. If necessary, in order to block the external environment during implantation, the PE ring may be attached to the wound site and then vascular progenitor cells may be implanted therein.

In addition, the present invention differentiates embryonic stem cells through the formation of embryoid bodies in a hypoxic state, isolating vascular progenitor cells, and then wound the mammal, characterized in that concentrated in high concentration through flow cytometry (FACS) using a marker Provided are embryonic stem cell-derived vascular progenitor cells.

Human embryonic stem cells may be used as the embryonic stem cells, and since these human embryonic stem cells have infinite proliferation, they can be continuously cultured through passage culture, and can be distributed by a known method such as freeze drying. Can be provided as a commodity.

In addition, the present invention is to differentiate the embryonic stem cells through the formation of embryoid body in the hypoxic state to separate the vascular progenitor cells, and then to include the embryonic stem cell-derived vascular precursor cells concentrated at high concentration through flow cytometry (FACS) using a marker It provides an implant for the treatment of wounds. The embryonic stem cells may be used human embryonic stem cells, it can be provided as a commodity that can be distributed by adding a preservative, a culture solution, etc. to these human embryonic stem cells.

In the wound treatment method of the present invention, by using human embryonic stem cells having infinite self-proliferation ability as a cell source, not only can greatly reduce the problem of cell supply restriction, but also adult stem cell-derived vascular precursors such as conventional cord blood stem cells. It is effective to treat the wound site more efficiently in a short time than using cells.

In particular, the progress after transplantation of human embryonic stem cell-derived vascular progenitor cells of the present invention shows that the collagen layer is formed on the wound site much more effectively than the comparative group such as umbilical cord blood stem cell-derived vascular progenitor cells, thereby promoting re-epithelialization more effectively. Able to know. In addition, according to the treatment method of the present invention, new blood vessels generated by transplanted human embryonic stem cell-derived vascular precursor cells and angiogenesis-promoting factors secreted therefrom are wound healing factors (growth factors, inflammatory cells) present in the animal body. , Stromal cells and skin progenitor cells, etc.) can be made more effective in treating wounds.

Therefore, the wound treatment method of the present invention has no limitation of cell supply, and can heal wounds in a shorter time and with higher efficiency than conventional adult stem cell wound treatment methods.

As used herein, "embryonic stem cells" include all embryonic stem cells derived from mammals, and include human embryonic stem cells.

The human embryonic stem cells comprise pluripotent cells derived from the inner cell mass of human morula. The human embryonic stem cells are, for example, CHA-hES3 (Jumi Kim, Sung-Hwan Moon, Soo-Hong Lee, Dong-Ryul Lee, Gou-Young Koh, and Hyung-Min Chung, Effective Isolation and Culture of Endothelial Cellsin Embryoid Body Differentiated from Human Embryonic Stem cells (2007) STEM CELLS AND DEVELOPMENT 16: 269.280) and the like may be used, but is not limited to these examples. In addition, human embryonic stem cells can be easily constructed by those skilled in the art.

In the present invention, "human embryonic stem cell-derived vascular progenitor cells" refers to vascular progenitor cells derived from human embryonic stem cells and include high concentrations of pure vascular progenitor cells from which teratoma has been removed.

"Human embryonic stem cell-derived vascular progenitor cells" include vascular progenitor cells concentrated at high concentration through flow cytometry after differentiating human embryonic stem cells through embryoid formation in a hypoxic state.

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One.

(1) Culture and proliferation of human embryonic stem cell-derived vascular progenitor cells

Human embryonic stem cells were differentiated through embryoid formation in a hypoxic state, and human embryonic stem cell-derived vascular progenitor cells were separated by high concentration by flow cytometry using a double marker of CD133 / KDR.

When the human embryonic stem cell-derived vascular progenitor cells from which the teratoma was removed were cultured in an EGM-2 / MV (Cambrex) culture on a collagen-coated culture dish, and the concentration of cells was distributed in the culture dish at about 70-80%. Proliferation was carried out using a trypsin-EDTA (Gibco) from the culture dish and subcultured.

(2) creation of wound animal models

The wound animal model for the verification of wound treatment effect was induced by using a biopsy punch on the back of a 6-week-old nude mouse to induce a 12 mm delamination of the skin, and then excluding the treatment effect due to the natural healing ability. PE ring was mounted on the wound site.

The wound animal model is manufactured as shown in FIG. 2.

(3) Transplantation of Human Embryonic Stem Cell-Derived Vascular Progenitor Cells into a Wound Animal Model

The cultured human embryonic stem cell-derived vascular progenitor cells were transplanted into the prepared wound animal model. At the time of transplantation, 3 × 10 ⁵ human embryonic stem cell-derived vascular progenitor cells were mixed with 200 μl of matrigel and transplanted into the PE ring of the wound animal model.

In order to verify the efficacy of human embryonic stem cell-derived vascular progenitor cells, the culture group (EGM-2 MV medium, 200 μl), human fibroblasts (hEF, Fibroblast, 3 × 10 ⁵ ), cord blood-derived blood vessels were compared to the same wound animal model. After implanting progenitor cells (CB-EPC, 3 × 10 ⁵ ), the extent of wound size reduction was observed for 2 weeks.

Two weeks later, histological analysis and immunostaining showed the synthesis and re-epithelialization of collagen, an important factor for wound healing.

Collagen synthesis was confirmed by Masson's Trichrome staining, and re-epithelialization was confirmed by CK6 staining.

Experimental Example  One.

Human embryonic stem cell-derived vascular progenitor cells cultured under the conditions specified in Example (1) of Example 1 were observed morphologically. As shown in Figure 1a it was confirmed that the morphological characteristics similar to the cord blood-derived vascular precursor cells (CB-EPC) to be used as a positive control. In order to verify the characteristics of the human embryonic stem cell-derived vascular progenitor cells as vascular progenitor cells with morphological analysis, the human embryonic stem cell-derived vascular progenitor cells passaged three times as shown in FIG. Expression of CD34, KDR, SMA, Flt-1, Tie-2, vWF, and the like as cell markers was confirmed.

The results indicate that human embryonic stem cell-derived vascular progenitor cells used in the present invention have characteristics similar to those of conventional vascular progenitor cells.

Experimental Example  2.

After the wound animal model was prepared by the method specified in Example (2), human embryonic stem cell-derived vascular progenitor cells and other comparative groups were mixed with matrigel and implanted into the PE ring on the back of the wound animal model (see FIG. 2). ).

Experimental Example  3.

As specified in Example (3), culture medium (EGM-2 MV medium, 200 μl), human fibroblasts (hEF, Fibroblast, 3 × 10 ⁵ ), cord blood-derived vascular precursor cells (CB-EPC, 3 × 10 ⁵ ), human embryonic stem cell-derived vascular progenitor cells (hES-VAPC, VAPC, 3 × 10 ⁵ ) were transplanted, and each treatment group was observed for 2 weeks to observe the extent of wound size reduction (see FIG. 3). As shown in FIG. 3, in the medium treated group (Medium), the wound size was decreased most recently, and in the case of human fibroblast and umbilical cord blood-derived vascular progenitor cell treated group (CB-EPC), somewhat faster than the medium treated group. Although the wound size was decreased, the wound size of the human embryonic stem cell-derived vascular progenitor cell treatment group (VAPC) was significantly faster than that of the other treatment groups.

The results indicate that the effect of wound treatment using human embryonic stem cell-derived vascular progenitor cells used in the present invention is significantly more efficient than other treatment groups.

Experimental Example  4.

As indicated in (3) of Example 1, human embryonic stem cell-derived vascular progenitor cells were transplanted into the wound animal model, and two weeks later, samples of the wound region were taken and histologically analyzed.

As can be seen in Figure 4a, in the case of human embryonic stem cell-derived vascular progenitor cell transplant group treated according to the treatment method of the present invention, the synthesis of collagen was significantly higher than the comparison group culture treatment group. CK6 immunostaining of the same tissue specimens confirmed that re-epithelialization occurred faster than the culture solution treatment group (see FIG. 4b).

The above results indicate that the wound healing effect using human embryonic stem cell-derived vascular progenitor cells used in the present invention is a large amount of collagen synthesis essential for wound healing, which accelerated re-epithelialization by performing a crosslinking role for re-epithelialization.

Experimental Example  5.

As specified in Example 1 (3), human embryonic stem cell-derived vascular progenitor cells were transplanted into the wound animal model, and after 2 weeks, samples of the wound region were taken and subjected to fluorescence immunostaining of tissue specimens.

Human embryonic stem cell-derived vascular progenitor cells were labeled with DiI (red) and transplanted for identification of transplanted cells by fluorescence immunostaining method. After injection through the tail, tissue was collected and confirmed by fluorescence microscopy.

As can be seen in Figure 5, in the case of transplanting human embryonic stem cell-derived vascular progenitor cells, human embryonic stem cell-derived vascular precursor cells labeled with DiI (red) at the site where Lectin (green) is expressed It confirmed that it exists.

The results indicate that the human embryonic stem cell-derived vascular progenitor cells used in the present invention generated new blood vessels in the wound site after transplantation, and thus, it would be easier to transfer wound healing factors to the wound site, thereby enabling more effective wound healing. It means that the effect appeared.

Figure 1a is a photograph showing the morphological appearance of human embryonic stem cell-derived vascular precursor cells, Figure 1b is a representative vascular cell markers of human embryonic stem cell-derived vascular precursor cells CD34, KDR, Flt-1, SMA, Tie- 2, vWF expression was confirmed by flow cytometry.

Figure 2 is a photograph and a schematic diagram showing the wound animal model production and transplantation process for the verification of wound treatment effect.

Figure 3 is a culture medium (EGM-2 MV, 200ul), human fibroblasts (hEF, 3X10 ⁵ ), umbilical cord blood-derived vascular progenitor cells (CB-EPC, 3X10 ⁵ to verify the wound treatment effect of human embryonic stem cell-derived vascular precursor cells The wound size reduction was observed in the group transplanted with human embryonic stem cell-derived vascular progenitor cells (hES-VAPC, 3X10 ⁵ ) for 2 weeks.

Figure 4a is a result of confirming the synthesis of collagen through Masson's Trichrome staining after collecting the wound tissue two weeks later, Figure 4b is a result of confirming re-epithelialization through CK6 staining of the same tissue.

5 is a result of observing the degree of blood vessel generation in the same tissue as in Figure 4 through fluorescent immunostaining.

Claims (12)

(a) culturing vascular progenitor cells derived from human embryonic stem cells for transplantation; (B) a wound treatment method using the human embryonic stem cell-derived vascular precursor cells comprising the step of transplanting the cultured human embryonic stem cell-derived vascular progenitor cells to the skin of mammals other than humans. The method of claim 1, wherein the cell culture step is to differentiate human embryonic stem cells through the formation of embryoid bodies in a hypoxic state to separate vascular progenitor cells, and then concentrated human embryos concentrated by flow cytometry (FACS) using a marker. Wound treatment method using human embryonic stem cell-derived vascular precursor cells, which are human embryonic stem cells using stem cell-derived vascular precursor cells. delete The method for treating wounds using human embryonic stem cell-derived vascular precursor cells according to claim 2, wherein the marker used for flow cytometry is a double marker of CD133 / KDR. According to claim 1 or 2, wherein the culture step is to culture the human embryonic stem cell-derived vascular progenitor cells with the culture after the collagen coating on the culture dish, wound using human embryonic stem cell-derived vascular precursor cells Method of treatment. The wound treatment method according to claim 5, wherein the culture medium is EGM-2 MV culture medium, trypsin-EDTA, DMEM culture medium, M199 culture medium, or a mixed culture thereof. The method of claim 1, wherein the human embryonic stem cell-derived vascular progenitor cells are transplanted with matrigel (matrigel) to form a support for the cells to be transplanted, wound using human embryonic stem cell-derived vascular precursor cells Method of treatment. The method of claim 1 or 7, wherein the implantation step is to install a blood ring (PE) in the wound site and then implant the vascular precursor cells therein, wound treatment using human embryonic stem cell-derived vascular precursor cells Way. delete delete Human embryonic stem cells are differentiated through the formation of embryoid bodies in a hypoxic state to separate vascular progenitor cells, and then wound treatment comprising highly concentrated human embryonic stem cell-derived vascular progenitor cells through flow cytometry (FACS) using markers. Implants for. delete

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