KR20130013435A - Process for the proliferation of placenta-derived stem cells - Google Patents

Abstract

PURPOSE: A method for proliferating placenta-derived stem cells is provided to suppress stem cell differentiation and to maintain stemness. CONSTITUTION: A method for proliferating placenta-derived stem cells comprises a step of sub-culturing the placenta-derived stem cells under a hypoxic condition. The stem cells are amniotic mesenchymal stem cells which are derived from amnion of human placenta or amniotic epithelial cells of human placenta. The sub-culture is performed in an alpha-MEM medium containing 10% FBS, 1% of penicillin-streptomycin, 1ug/ml of heparin, and 25 ng/ml of FGF-4(Fibroblast Growth Factor-4).

Description

Process for the proliferation of placenta-derived stem cells

The present invention relates to a method for propagation of placental-derived stem cells, and more particularly, to placenta-derived stem cells comprising subcultured under hypoxia conditions having an oxygen partial pressure of 1 to 8%. It relates to a method of proliferating stem cells.

By differentiating stem cells such as embryonic stem cells and adult stem cells into various cells, various studies have been made on the possibility of application as cell therapy. Embryonic stem cells with multipotency have been noted as cell therapy by differentiating into various cells, but due to ethical problems in utilizing embryonic stem cells, they have difficulty in practical use as cell therapy. In order to circumvent such ethical problems, studies using adult stem cells have been actively conducted. As adult stem cells, stem cells derived from bone marrow, adipose, umbilical cord blood, etc. are known. Various studies have been conducted on how to differentiate these cells into specific cells.

For the development of cell therapy products using adult stem cells without ethical problems, it is essential to establish a method that can effectively proliferate while maintaining the stem stem cell (stemness) of the cells. However, adult stem cells have a low proliferation rate and are easily aged and have a limited number of cells that can be obtained from a tissue. In general, bone marrow-derived adult stem cells are known to have extremely limited differentiation into various tissues and are difficult to obtain a large number of cells (Matikainen T. and Laine J. Placenta-an alternative source of stem cells.Toxicology and Applied Pharmacology 2005; 207: S544-S549). Other types of adult stem cells, umbilical cord blood or fat stem cells, are relatively easy to obtain, but have limited differentiation capacity. Placent-derived stem cells, which are of recent interest, have the advantage that they do not require invasive procedures compared to other adult stem cells, but the number of cells from one placenta is also limited, and the number of cells can be expanded. Even if the subculture is carried out for risk, the period of proliferation of the cells is reduced, and thus the number of cells eventually obtained is intact.

In addition, adult stem cells have different factors on stem cell maintenance, aging mechanism, and proliferation, depending on the source. For example, when adipose-derived stem cells and bone marrow-derived stem cells are cultured under the same or similar conditions, differentiation may be induced or differentiation may be suppressed depending on the type of stem cells. In addition, in the differentiation induction, the types of cells differentiated and produced according to the types of stem cells may appear at all. For example, when preadipocytes are exposed to hormones that induce differentiation into adipocytes, they differentiate into adipocytes.However, in hypoxia conditions, progenitor cells are exposed to hormones that induce differentiation of adipocytes. It has been reported that they do not differentiate into adipocytes (Lin Q, Lee YJ, Yun Z. Differentiation arrest by hypoxia. J Biol Chem. 2006 Oct 13; 281 (41): 30678-83. Epub 2006 Aug 22). However, it has also been reported that when bone marrow-derived mesenchymal stem cells are cultured in hypoxia conditions, the differentiation ability of adipocytes and osteoblasts is increased (Grayson WL, Zhao F, Izadpanah R, Bunnell B). , Ma T: Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs J cell Physiol 2006, 207:. 331-339; Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status. BMC Cell Biology 2011, 12: 12; Song WW, Bai H, Wang CB, Yu LL, Ou JF, Zhao Q, Su YN. Effects of hypoxia on the proliferation of human bone marrow mesenchymal stem cells Zhonghua Yi Xue Za Zhi. 2010 Aug 10; 90 (30): 2149-52).

Therefore, in order to develop a cell therapeutic agent using adult stem cells, it is required to develop a method capable of effectively proliferating while maintaining stem cell nature (stemness) and inhibiting aging to the specific adult stem cells.

The present inventors conducted various studies to develop a method capable of proliferating at high proliferation rate while maintaining stem stemity of placental-derived adult stem cells and also inhibiting aging as much as possible. As a result, when culturing placenta-derived adult stem cells under hypoxic conditions with an oxygen partial pressure of 1 to 8% (specifically, passage), the differentiation of placental-derived adult stem cells is not only suppressed but also high proliferation rate. It was found that can be achieved. This is surprising given the reports that many stem cells disclose that differentiation is induced under hypoxic conditions.

Accordingly, an object of the present invention is to provide a method for propagating placental-derived stem cells, including culturing placental-derived adult stem cells under hypoxic conditions having an oxygen partial pressure of 1 to 8%.

According to one aspect of the present invention, there is provided a method for proliferation of placental-derived stem cells, comprising subcultured placenta-derived stem cells under low oxygen conditions having an oxygen partial pressure of 1 to 8%.

In the method for propagating placental-derived stem cells according to the present invention, the placental-derived stem cells are derived from amniotic mesenchymal stem cells derived from amnion of human placenta or isolated human placenta. Amniotic epithelial cells can be preferably used.

In addition, the passage may be preferably performed up to 10 to 15 passages.

In the method of propagation of placental-derived stem cells according to the present invention, the subculture is performed in alpha-MEM medium to which fetal bovine serum, antibiotic, heparin, and fibroblast growth factor-4 are added. In one embodiment, the subculture is alpha-MEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 ug / ml heparin, 25 ng / ml fibroblast growth factor-4. It can be carried out in the.

According to the present invention, when subcultured placenta-derived adult stem cells under hypoxia conditions having an oxygen partial pressure of 1 to 8%, stem cell differentiation can be suppressed to maintain stem cellity (stemness). In addition, aging can be suppressed as much as possible. In particular, the proliferation method according to the present invention can achieve a significantly higher proliferation rate compared to normoxia conditions. Therefore, the proliferation method of placental-derived stem cells according to the present invention can shorten the culture rate of the cells, thereby enabling mass production of placental-derived stem cells.

1A and 1B show passage of mesenchymal stem cells (hAMSC) derived from amnion of human placenta (FIG. 1A) and amnion epithelial cells (hAEC) of human placenta (FIG. 1B), respectively, under normal oxygen and hypoxic conditions. When the shape of the stem cells is observed under an optical microscope.
2A to 2C show the cumulative number of cells obtained when subcultured hAMSC under normal oxygen conditions and low oxygen conditions (FIG. 2A), the ratio of cell numbers under low oxygen conditions to normal oxygen conditions of the number of accumulated cells obtained. (FIG. 2B) and the results of measuring the doubling time of the cells (FIG. 2C).
3A to 3C show the cumulative number of cells obtained when passage of hAEC under normal oxygen conditions and low oxygen conditions (FIG. 3A), the ratio of cell numbers under low oxygen conditions to normal oxygen conditions of the number of accumulated cells obtained. (FIG. 3B), and the result of measuring the Population doubling Level (PDL) (FIG. 3C).
Figure 4 shows the results of analyzing the surface antigen of the proliferated cells when the hAMSC was passaged under normal oxygen conditions and low oxygen conditions using a flow cytometry.
Figure 5 shows the results of analyzing the surface antigen of the proliferated cells when the hAEC was passaged under normal oxygen conditions and low oxygen conditions using a flow cytometer.
6A and 6B show the results of staining analysis of stem cell markers (FIG. 6A) and proliferation markers (FIG. 6B) of proliferated cells when hAMSCs were passaged under normal oxygen and hypoxic conditions.
7A and 7B show the results of staining analysis of stem cell markers (FIG. 7A) and proliferation markers (FIG. 7B) of proliferated cells when hAEC was passaged under normal oxygen and hypoxic conditions.
8 shows the expression of Oct4, Sox2, nanog, c-Myc, KLF4 using reverse transcription polymerase chain reaction (RT-PCR) for harvested cells when hAMSC was passaged under normal oxygen and hypoxic conditions. One result is shown.

As used herein, the term "placenta-derived stem cells" includes all stem cells isolated from the placenta, and preferably, four types of stem cells isolated from the human placenta separated in vitro. That is, (1) human amniotic epithelial cells (hAEC), (2) amniotic mesenchymal stromal cells or human amniotic mesenchymal stem cells (hAMSC), and 3) chorionic mesenchymal stem cells. Cells (human chorionic mesenchymal stromal cells or human chorionic mesenchymal stem cells (hCMSC)), and (4) human chorionic trophoblastic cells (hCTC). More preferably, the placental-derived stem cells may be mesenchymal stem cells (hAMSC) derived from the amnion of the human placenta separated in vitro or amniotic epithelial cells (hAEC) of the human placenta separated in vitro. The placental-derived stem cells can be obtained by known methods (eg, Current protocols in Stem Cell Biology 1E.3.1-1E.3.10 and 1E.5.1-1E5.11).

The present invention provides a method for propagating placental-derived stem cells, comprising subcultured placenta-derived stem cells under low oxygen conditions having an oxygen partial pressure of 1-8%. When the placenta-derived adult stem cells are passaged under hypoxia conditions as described above, stem cell differentiation can be suppressed to maintain stem cellity and to suppress aging as much as possible. In particular, it has been found that significantly higher proliferation rates can be achieved compared to normoxia conditions.

In the proliferation method of placental-derived stem cells according to the present invention, the passage number of the passage is not particularly limited, and the passage number may be appropriately selected according to the number of desired proliferating cells. Typically, at least 5 passages, preferably 10 passages or more, more preferably 10 to 15 passages, can obtain a clinically necessary number of cumulative proliferating cells.

The growth media used in the method for propagating placental-derived stem cells according to the present invention are not particularly limited, and preferably, fetal bovine serum, antibiotics, heparin, and fibroblast growth factor-4 -4) can be performed in the added alpha-MEM medium. In one embodiment, the subculture may be performed in alpha-MEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1 ug / ml heparin, and 25 ng / ml fibroblast growth factor-4. have.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples. However, these Examples and Test Examples are for illustrative purposes only, and the scope of the present invention is not limited to these Examples and Test Examples.

Example  1. Proliferation of Placental-Derived Stem Cells

(1) derived from the amnion of the placenta Intermediate lobe  Stem Cells( human amniotic mesenchymal  stem cells , hAMSCs Proliferation of

Mesenchymal stem cells (hAMSCs) obtained from the amniotic membrane of human placenta were subjected to normal oxygen conditions (ie, 37 ° C, 5% CO ₂ culture conditions and hypoxia conditions, 37 ° C, 5% CO ₂ , 1 ～). The cells were cultured in a culture condition of 8% O ₂ . Expansion media include 10% fetal bovine serum, 1% penicillin-streptomycin, 1ug / ml heparin, 25ng / ml fibroblast growth factor-4 (FGF4). Added alpha-MEM medium was used. hAMSCs were inoculated by counting 6000 to 7000 cells per cm ² , and cultured with changing medium every other day. More than 80% of the cells were grown (confluent) in the flask for about 3 to 4 days after incubation. The cultured cells were washed with phosphate-buffered saline (PBS) and then washed for 2 minutes using 0.25% trypsin / EDTA. After enzymatic treatment, fetal bovine serum was added to stop the enzyme reaction, centrifuged at 1000 rpm for about 5 minutes, and then the supernatant was removed to harvest the cells. Harvested cells were again counted to 6000-7000 cells per cm ² and subjected to the same method of subculture.

(2) amnion epithelial cells of the placenta ( human amniotic epithelial cells , hAECs Proliferation of

Human placenta amnion epithelial cells (hAECs) were subjected to passage under normal oxygen conditions (hymoxia conditions) and hypoxia conditions under the same conditions as in (1) above.

Test Example  1. Normal oxygen condition and Hypoxia  Comparison of Morphology of Stem Cells Cultured in Conditions

According to Example 1 (1), the results of observing the passaged hAMSCs (first passage, fifth passage, and tenth passage) under normal oxygen conditions and low oxygen conditions, respectively, with an optical microscope are shown in FIG. 1A. From the results of FIG. 1A, it can be seen that cells cultured under normal oxygen conditions and low oxygen conditions were passaged while maintaining the morphology of mesenchymal cells.

FIG. 1B is an optical microscope of hAECs cultured to the 0th passage, the 1st passage, the 5th passage, the 10th passage, and the 15th passage, respectively, in the normal oxygen condition and the low oxygen condition in Example 1 (2). The result is.

Changes in cell morphology were observed after passage 1 during passage, but the shape was maintained and no difference in cell morphology between normal and hypoxic conditions was observed. However, more cell numbers were observed in hypoxic conditions than in normal oxygen conditions.

Test Example  2. By subculture  Number of accumulated cells obtained

When the hAMSCs were passaged to the 14th passage under normal oxygen conditions and low oxygen conditions according to Example 1 (1), the results of cumulative counting of the proliferated cells are shown in FIG. 2A. In addition, the results showing the number of cells proliferated in each passage (the number of accumulated cells) as the ratio of cell numbers of hypoxic conditions to normal oxygen conditions is shown in Figure 2b. 2A and 2B, when cultured to the 10th passage, the number of cells proliferated in the normal oxygen condition was only about 1.6x10 ¹³ , but the number of cells proliferated in the low oxygen condition was about 5x10 ¹⁵ cells. When the cells were cultured up to the 10th passage, the cumulative number of cells showed a proliferation rate of about 6000 times higher than that in the normal oxygen condition. In addition, the level of doubling of cells under each condition (PDL) was measured, and the sum of doubling levels up to the 11th passage was normal oxygen condition of 20 and low oxygen condition of 29.5. At all times, the doubling levels of hypoxic conditions remained high. Particularly, the third passage showed the highest value of 3.5, the low oxygen condition of the 11th passage was low value of 1.5, and the normal condition rapidly decreased after the 9th passage, and cell growth could be stopped. Particularly, in the eleventh passage, it can be estimated that the cell death starts by recording a value of -0.5 (see FIG. 2C). From the above results, it can be seen that the hypoxic culture conditions increase the proliferation rate of the cells and also delay the aging of the cells.

When hAEC was passaged to the 15th passage under normal oxygen conditions and low oxygen conditions, respectively, according to Example 1 (2), the number of cells proliferated in the same manner as described above and the number of cells proliferated in each passage were normal oxygen. The results shown in the cell number ratio of the hypoxic condition to the condition, and the result of measuring the doubling time are as shown in Figures 3a, 3b and 3c, respectively. When cultured to the 15th passage, the number of cells proliferated under normal oxygen conditions was only about 6x10 ¹¹ , but the number of cells proliferated under low oxygen conditions was about 1.3 x 10 ¹⁵ , and the cumulative number of cells was When cultured to the 15th passage, the culture rate in the hypoxic condition showed a growth rate of about 2000 times or more than that in the normal oxygen condition.

In addition, as a result of measuring the doubling level (PDL) of the cells under each condition, the sum of the doubling levels up to the 10th passage was 10.8 normal oxygen condition, 21.9 hypoxic condition and approximately 2 times higher hypoxic condition. At all stages of culture, doubling levels of hypoxic conditions remained high. In particular, under normal conditions, the doubled level (PDL) level was below 1.5, while in low oxygen conditions, it gradually increased to 2 or more after the second pass, and reached the maximum value of 4 in the fourth pass, and 2 to the 9th pass. It stayed above. From the above results, as in hAMSCs, it can be seen that when hAEC is cultured under low oxygen, cell proliferation is increased and cell aging is delayed.

Test Example  3. Characterization of Stem Cells in Normal and Low Oxygen Environments

(One) Surface antigen  analysis - Flow cell  analysis

According to (1) of Example 1, the surface antigens of cells harvested by subcultured hAMSCs to the first passage, the fifth passage, and the tenth passage under normal oxygen conditions and low oxygen conditions were analyzed. That is, each harvested cells were washed with a mixture of 5% FBS and PBS, and then centrifuged at 1000 rpm for 5 minutes. After the supernatant was discarded, the cells were suspended in FACS buffer and 10000 cells were divided by the number of samples. In each well, add antibodies (FITC-labeled anti-SSEA4, anti-TRA-1-81, anti-CD34 and PE-labeled anti-TRA-1-60, anti-CD9, anti-CD44), respectively. After reacting for 20 minutes at ℃ was analyzed using a flow cytometer (flow cytometery), the results are as shown in FIG. From the results of FIG. 4, it can be seen that the MSC marker CD44 is maintained up to the 10th passage in cells cultured under normal oxygen and hypoxic conditions, so that the characteristics of mesenchymal cells are not changed. The stem cell marker SSEA4 decreased with passage, but the decrease was lower in the cells cultured in hypoxic conditions than in cells cultured in normal oxygen conditions.

In addition, according to Example 1 (2), the surface of cells harvested by subcultured hAEC to 0th passage, 1st passage, 5th passage, 10th passage, and 15th passage under normal oxygen conditions and low oxygen conditions, respectively Antigen was analyzed in the same manner as above. SSEA4, TRA-1-60, and TRA-1-81 as a stem cell marker, CD9 as a nontropoblast marker, CD34 as a hematopoetic stem cell marker, CD15 as a NPC marker, CD 133, And CD 184, CD 44 and CD90 as MSC markers, respectively. The result is shown in FIG. From the results of FIG. 5, no significant difference was found in cells cultured under normal and low oxygen conditions.

(2) Marker  Protein staining analysis

According to (1) of Example 1, staining analysis of marker proteins of cells harvested by subcultured hAMSCs to the first passage, the fifth passage, and the tenth passage under normal oxygen conditions and low oxygen conditions, respectively, was performed. That is, each harvested cell was washed three times with PBS and fixed for 10 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, and treated with a blocking buffer (5% goat serum) containing 0.3% Triton-X100 for 1 hour at room temperature, the stem cell markers Oct4 and Sox2 in blocking buffer 1 Dilute to 400 and react overnight at 4 ° C. Washed three times with PBS, and reacted with secondary antibodies Alexa Fluor ^TM 488 and Alexa Fluor ^TM 594 in the dark for 1 hour. After washing three times with PBS, it was placed on a slide glass and observed by fluorescence microscopy. As a result, Sox2 was expressed while Oct4 was not expressed in both normal oxygen condition and hypoxic condition (see FIG. 6A). In addition, as a result of staining analysis for Ki-67, which is a proliferation marker, cells were normally growing (see FIG. 6B). In particular, as can be seen from the results of FIG. 6B, the expression level of Ki-67 was high in cells cultured under hypoxic conditions, and the proliferation rate was significantly high.

In addition, according to Example 1 (2), the markers of cells harvested by subcultured hAEC to 0th passage, 1st passage, 5th passage, 10th passage, and 15th passage under normal oxygen conditions and low oxygen conditions, respectively Was analyzed in the same manner as above. From the results of FIGS. 7A and 7B, as in hAMSCs, Oct4 was not expressed in both normal oxygen and hypoxic conditions, but Sox2 was expressed, and the expression level of Ki-67, a proliferation marker, was also high in cells cultured in hypoxic conditions. It can be seen that the proliferation rate is significantly high.

(3) Marker  Protein Reverse transcription Polymerization Chain Reaction  analysis

Reverse transcription polymerase chain reaction (RT-PCR) on cells harvested by passage of hAMSCs to the first passage, the fifth passage, and the tenth passage under normal oxygen conditions and low oxygen conditions, respectively, according to Example 1 (1). Expression of Oct4, Sox2, nanog, c-Myc, KLF4 was confirmed using. RT-reaction was carried out at 42 ° C for 60 minutes, at 70 ° C for 10 minutes, and PCR-reaction was carried out for 30 to 35 times at 95 ° C for 5 minutes and at 95 ° C for 35 seconds / 60 ° C for 45 seconds / 72 ° C for 20 seconds. Cycles were performed, followed by 10 minutes at 72 ° C., followed by electrophoresis on 2% agarose. Primers used in the PCR-reaction are shown in Table 1 below.

Gene name SEQ ID NO: primer product size Oct3 / 4 One sense 5'-ggacaggggaggggaggagctagg-3 ' 143bp 2 Antisense 5'-cttccctccaaccagttgccccaaac-3 ' Sox2 3 sense 5'-gccgagtggaaacttttgtc-3 ' 264 bp 4 Antisense 5'-gttcatgtgcgcgtaactgt-3 ' Nanog 5 sense 5'-ttccttcctccatggatctg-3 ' 206 bp 6 Antisense 5'-tctgctggaggctgaggtat-3 ' C-MYC 7 sense 5'-tacatcctgtccgtccaagca-3 ' 300bp 8 Antisense 5'-tcagccaaggttgtgaggttg-3 ' KLF4 9 sense 5'-cccacacaggtgagaaacct-3 ' 169 bp 10 Antisense 5'-atgtgtaaggcgaggtggtc-3 '

The reverse transcription polymerase chain reaction (RT-PCR) analysis results are shown in FIG. 8. From the results of Figure 8, except for cMyc stem cell marker genes (Oct4, Nanog, Klf4, Sox2) expression was reduced over passage. However, the reduction pattern was apparent in cells cultured under normal oxygen conditions, and in cells cultured under hypoxic conditions, there was a difference in expression of Sox2 in the fifth passage and Nanog in the tenth passage, and other genes showed a significant difference. Did. From these results, it can be seen that the cells cultured in the hypoxic condition not only have a high proliferation rate but also maintain the characteristics of placental-derived stem cells for a longer time than the cells cultured in the normal oxygen condition.

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Claims (5)

A method of propagating placental-derived stem cells, comprising subcultured placental-derived stem cells under hypoxic conditions with an oxygen partial pressure of 1-8%. The method of claim 1, wherein the placental-derived stem cells are derived from amniotic membranes of human placenta (amniotic mesenchymal stem cells) or human placenta (amniotic epithelial cells) isolated in vitro Proliferation of placenta-derived stem cells, characterized in that the. The method of claim 1, wherein the subculture is performed up to 10 to 15 passages. The subculture according to any one of claims 1 to 3, wherein the subculture is performed in alpha-MEM medium to which fetal bovine serum, antibiotics, heparin, and fibroblast growth factor-4 are added. Method for proliferation of placenta-derived stem cells, characterized in that. The method of claim 4, wherein the subculture is performed in alpha-MEM medium to which 10% fetal bovine serum, 1% penicillin-streptomycin, 1 ug / ml heparin, and 25 ng / ml fibroblast growth factor-4 are added. Method for proliferation of placenta-derived stem cells, characterized in that.

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