KR101833960B1 - A method of Effects of three dimensional culture of spermatogonial stem cell and use of the same - Google Patents
A method of Effects of three dimensional culture of spermatogonial stem cell and use of the same Download PDFInfo
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Abstract
The present invention relates to a three-dimensional in vitro culture method for spermatogonial stem cells, and more particularly to a method for culturing spermatogonial stem cells in a three-dimensional hydrogel based on an agarose-based three-dimensional hydrogel or a polyethylene glycol (PEG) To maintain self regeneration and undifferentiation of porcine spermatogonial stem cells and their use. As can be seen through the present invention, agarose and PEG-based 3D hydrogels can be used as powerful systems for the maintenance of spermatogonial stem cells.
Description
The present invention relates to a three-dimensional in vitro culture method of spermatogonial stem cells and uses thereof.
Spermatogonial stem cells (SSCs) derived from primordial germ cells regenerate endlessly in the basal compartment and differentiate into sperm in the adluminal compartment of the tubule. Their pluripotency can also be induced by specialized microenvironments (Kostereva N1, Hofmann MC. Reprod Domest Anim. 2008 Jul; 43 Suppl 2: 386-92. Doi: 10.1111 / j.1439-0531.2008 .011894). Thus, they have been used to treat male infertility (Kubota H1, Brinster RL.Nat Clin Pract Endocrinol Metab 2006 Feb; 2 (2): 99-108), transgenic sperm production (Kanatsu-Shinohara M1, Toyokuni S, Shinohara T. Biol Reprod. 2005 Jan; 72 (1): 236-40. Epub 2004 Sep 29) and development of male patient-specific cell-based therapies (Rao M1, Condic ML, Stem Cells Dev. 10. doi: 10.1089 / scd.2008.0013).
In general, cell culture systems consist of physico-chemical, physiological and ECM (extracellular matrix) niche.
Thus, up to now, in vitro culture methods that support self-renewal maintenance in SSCs are physiochemical (Antoni D, Burckel H, Josset E, Noel G. Int J Mol Sci., 2015 Mar 11; 16 (3): 5517-27. doi: 10.3390 / ijms16035517. Review: Lee WY1, Park HJ, Lee R, Lee KH, Kim YH, Ryu BY, Kim JH, Kim JH, Moon SH, Park JK, Chung HJ, Kim DH, (Anjamrooz SH1, Movahedin M, Tiraihi T, Mowla SJ, Reprod Fertil, < RTI ID = 0.0 > (2004), pp. 183-197 (2007), pp. 183-186, 1987. This paper focuses on the development of NISS, This confronted their great limitation in stem cell fate control (Ebata KT1, Yeh JR, Zhang X, Nagano MC. Exp Cell Res. 2011 Jun 10; 317 (10): 1319-29. Doi: 10.1016 / j.yexcr. Mar 23, 2011. Epub 2011 Mar 21). In other words, the absence of effective SSC culture for long-term cultivation has not been solved, and a great deal of interest in ECM Nish has been amplified.
To date, the self-renewal of SSCs has been implicated in various feeder cell-derived cellular niches (Nagano M1, Avarbock MR, Leonida EB, Brinster CJ, Brinster RL. Tissue Cell 1998 Aug; 30 (4): 389-97; , Brinster CJ, Avarbock MR, Brinster RL, Biol Reprod 2003 Jun; 68 (6): 2207-14.Apub 2003 Jan 22; Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Oguraya, Shinohara T. Biol Reprod 2005 Apr; 72 (4): 985-91, Epub 2004 Dec 15) and purified ECM protein-derived non-cells (Akbarinejad V, Tajik P, Movahedin M, Youssefi R, Shafiei S, Mazaheri Z. Anim Reprod Sci. 2015 Jun; 157: 95-102. Doi: 10.1016 / j.anireprosci.2015.04.003. Epub 2015 Apr 15; Kim BG1, Cho CM, Lee YA, Kim BJ, Kim KJ, Kim YH, Min KS , Kim CG, Ryu BY Biol Reprod 2010 Jun; 82 (6): 1162-9 doi: 10.1095 / biolreprod.109.079558 Epub 2010 Feb 10) It is maintained in a normal two-dimensional micro environment based on Nish.
However, the 2D culture system developed in the past does not show a powerful effect for a long time in promotion of SSC proliferation and inhibition of SSC differentiation (Kanatsu-Shinohara M, Toyokuni S, Shinohara T. Biol Reprod. -7 Epub 2004 Jun 9; Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Oguraya, Shinohara T. Biol Reprod 2005 Apr; 72 (4): 985-91. ) SSCs derived from large animals (cattle, pigs, horses, etc.) showed much difficulty in maintaining self-renewal even for a short time (Marret C1, Durand P. Reprod Nutr Dev. 2000 May-Jun; 19, Aponte PM1, Soda T, van de Kant HJ, de Rooij DG Theriogenology 2006 Jun; 65 (9): 1828-47). Therefore, as an alternative to solving these problems, interest has been focused on an in-
[Prior Patent Literature]
Korean Patent Publication No. 10-2005-0103270
DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and it is an object of the present invention to provide a culture method preferred by SSCs in maintenance of self-renewal.
In order to achieve the above object, the present invention provides a method for cultivating spermatogonial stem cells in an agarose-based three-dimensional hydrogel to maintain self regeneration of spermatogonial stem cells.
In one embodiment of the present invention, the amount of the agarose is preferably 0.2% (w / v), but is not limited thereto.
The present invention also provides a method for cultivating spermatogonial stem cells in an agarose-based three-dimensional hydrogel to maintain the undifferentiation of spermatogonial stem cells.
In one embodiment of the present invention, the amount of the agarose is preferably 0.2% (w / v), but is not limited thereto.
The present invention also provides a method for culturing spermatogonial stem cells in a polyethylene glycol (PEG) -based three-dimensional hydrogel to maintain the self-renewal of spermatogonial stem cells.
The present invention also provides a method for culturing spermatogonial stem cells in a polyethylene glycol (PEG) -based three-dimensional hydrogel to maintain the undifferentiation of spermatogonial stem cells.
The present invention also provides a composition for self regeneration maintenance of spermatogonial stem cells comprising an agarose-based three-dimensional hydrogel as an active ingredient.
In one embodiment of the present invention, the amount of the agarose is preferably 0.2% (w / v), but is not limited thereto.
The present invention also provides a composition for retaining undifferentiated spermatogonial stem cells comprising an agarose-based three-dimensional hydrogel as an active ingredient.
In one embodiment of the present invention, the amount of the agarose is preferably 0.2% (w / v), but is not limited thereto.
The present invention also provides a composition for self regenerating and maintaining spermatogonial stem cells comprising polyethylene glycol (PEG) -based three-dimensional hydrogel as an active ingredient.
The present invention also provides a composition for retaining undifferentiated spermatogonial stem cells comprising polyethylene glycol (PEG) -based three-dimensional hydrogel as an active ingredient.
In one embodiment of the present invention, the spermatogonial stem cell is preferably a porcine spermatogonial stem cell, but is not limited thereto.
Hereinafter, the present invention will be described.
The present invention is based on the finding that swine-derived SSCs having many economic characteristics are cultured in a three-dimensional scaffold without two-dimensional culture and ECM analogue in the presence or absence of feeder cells, and the survival rate, proliferation, morphology and SSC- Respectively.
Hereinafter, the present invention will be described in detail with reference to the drawings and the following description.
pig Of SSCs Three-dimensional for self-reproduction Agarose base Hydrogel Mechanism optimization
Three-dimensional scaffold agarose-based hydrogel system was used and the mechanism of 3-dimensional hydrogel supporting sufficient self-regeneration in porcine SSCs was cultured inside 3-dimensional hydrogel constructed by various activities of AP activity and agarose Were determined by measuring the colony size of the pig SSC. Although there is no significant difference between concentrations, the highest percentage of porcine SSCs with AP activity was observed in a 0.2% (w / v) Agarose-based 3D hydrogel (FIG. 1A). Also, the size of swine SSC-derived colonies of larger size than the 0.6% (w / v) Agarose-based 3D hydrogel was observed in a 0.2% (w / v) Agarose-based 3D hydrogel (FIG. These results indicate that the 0.2% (w / v) Agarose-based 3D hydrogel can effectively promote proliferation and differentiation in porcine SSCs and exhibit mechanical properties optimized for retention of porcine SSC self-renewal.
Effect of 3-D Culture Microenvironment on the Retention of Pig SSC Undifferentiation
Next, the usefulness of the three-dimensional culture microenvironment for the maintenance of porcine SSC undifferentiation was measured at transcriptional and translational regulation, AP activity, colony formation and morphological level of autoregulation specifically expressed in porcine SSCs. Older pig SSC colonies were uniformly formed 6 days after incubation in a 0.2% (w / v) Agarose-based 3D hydrogel and positively stained with strong red color by AP staining (Fig. 2C). On the other hand, porcine SSCs cultured in a two-dimensional microenvironment using STO cells as feeders (Fig. 2A) (Fig. 2B) did not form dome-shaped colonies and showed very weak positive AP staining . In addition, although in the 2D culture environment, the STO cell-derived cellular microenvironment provided a stronger retention of AP activity against porcine SSCs compared to the culture plate-derived non-petroleum microenvironment, and the AP activity Were detected in porcine SSCs cultured in 0.2% (w / v) Agarose-based 3D hydrogel (Figure 3). As shown in Figures 4 and 5, the same regulatory pattern between transcription and translation of self-renewing genes has been caused in the 3D culture microenvironment. Downregulation for significant transcription and translation of all self-renewing genes was not detected in porcine SSCs cultured in 0.2% (w / v) Agarose-based 3D hydrogel compared to 2D microenvironment. In other words, porcine SSCs cultured in a 0.2% (w / v) Agarose-based 3D hydrogel were NANOG , EPCAM and Transcription levels of UCHL1, and PLZF, OCT4, SOX2, and showed a marked increase significantly in the translation levels of TRA-1-81, although significant differences, even if not, OCT4 and THY1 , and NANOG And TRA-1-60. ≪ / RTI > From these results, we can see that, in the maintenance of undifferentiation, porcine SSCs prefer 3D environment rather than 2D incubation microenvironment.
Effect of 3D culture microenvironment on proliferation and survival of porcine SSCs
Finally, the effects of the 3D culture microenvironment on the survival and proliferative capacity of porcine SSCs were evaluated by measuring the ratio of colony diameter and viable cells after in vitro culture. Pig SSCs cultured in a 0.2% (w / v) Agarose-based 3D microenvironment, respectively, compared to a 2D microenvironment with or without STO cells, had the highest cell viability percent (Figure 6) and the largest colony diameter ). Moreover, in the 2D microenvironment, STO cell-derived cellular niche induced a much stronger survival of cell survival compared to the absence of cellular nissin (Fig. 6), and formation of small diameter colonies (FIG. 7), colony formation was not detected in a 2D microenvironment without cellular nissin derived from STO cells, suggesting that STO cell-derived cellular nisse is essential for maintaining the proliferation and survival rate of porcine SSCs. These results also suggest that the use of a 3D culture microenvironment in the culture of porcine SSCs is sufficiently effective to overcome the function of 2D STO cell-derived cellular niches in maintaining the proliferation and viability of porcine SSCs.
The present inventors investigated the survival rate (FIG. 2) and AP activity (FIG. 4) of SSCs cultured in 2D and 3D culture systems in order to compare characteristics according to dimensional differences. SSCs cultured on agarose-based 3D hydrogel showed the highest survival rate (100%), which was higher than that cultured in 2D environment. The AP activity of SSCs increased in 3D environment than 2D, suggesting that this effect affects the pluripotency of SSCs. In addition, the present inventors have compared the expression level of the molecular marker before multipotent markers such as OCT4, NANOG, SOX2, THY1, PLZF, UCHL1, EPCAM, TRA-1-60, TRA-1-81 required for the maintenance of the SSC. Expression levels (Figure 6) and translation (Figure 7) expression of SSC-specific markers were increased in the Agarose-based 3D environment versus the 2D environment.
As can be seen from the present invention, a 3D culture system using agarose and polyethylene glycol (HEG) -hydrogel can be used to maintain the expansion and self-renewal of SSCs, and AP activity is very high in this 3D system As well as being detected, increased mRNA and protein expression of SSC-specific genes was also observed in agarose and polyethylene glycol-based 3D hydrogels. Agarose and PEG-based 3D hydrogels are therefore a powerful system for the maintenance of SSCs.
Figure 1 shows the optimization of 3D hydrogel conditions to stimulate proliferation and maintain AP activity by incubating porcine SSC in agarose-based 3D hydrogel. The recovered pig SSCs were incubated for 6 days in a 3D hydrogel constructed by agarose at various concentrations (w / v), and the AP activity of the cultured porcine SSCs was calculated by AP staining to determine the percentage of cells stained positively And the proliferation was analyzed by measuring the colony diameter derived from porcine SSCs. In conclusion, pig SSCs cultured in 0.2% (w / v) Agarose-based 3D hydrogel had higher AP activity than 0.6% (w / v) Agaorose-based 3D hydrogel, although there was a significant difference between treatments Respectively. At the same time, pig SSC-derived colonies of significantly increased diameter were detected compared to 0.6% (w / v) Agarose-based 3D hydrogel in 0.2% (w / v) Agarose-based 3D hydrogel. All data represent the mean ± SEM of three independent experiments. * p < 0.05.
Degree Figure 2 shows AP activity of porcine SSC cultured in 2D and 3D microenvironments. The cultivation of undifferentiated porcine SSCs in a 3D microenvironment was carried out on a 0.2% (w / v) Agarose (w / v) medium, while the undifferentiation of porcine SSCs in the 2D microenvironment was maintained on the culture plate -based 3D hydrogel (C). Six days after incubation, cultured porcine SSCs were stained with AP and colony size was observed. AP staining (red) and larger colony sizes stronger than the 2D environment were detected in the 3D microenvironment. The scale bar is 50 μm.
Degree 6 shows the effect of 3D culture microenvironment on the survival rate of porcine SSCs. For 2D cultures, porcine SSCs were cultured in the presence or absence of STO cells. They were also encapsulated with 0.2% (wt / v) Agarose-based 3D hydrogel for incubation in 3D microenvironment of porcine SSCs. The cell viability of the cultured pSSCs was then determined by trypan blue exclusion assay on day 6 culture. The 3D microenvironment showed a significantly higher percentage of cell survival compared to the 2D microenvironment with or without STO cells. All data represent the mean ± SEM of four independent experiments. *, ** p < 0.05.
Figure 7 shows the effect of 3D culture microenvironment on proliferation of porcine SSCs. The undifferentiation of porcine SSCs in a 2D microenvironment was carried out in a 3D microenvironment within a 3D hydrogel constructed by 0.2% (w / v) Agarose, and maintained on 2D culture plates with or without STO cells coated. Cultivation in 3D microenvironment of undifferentiated porcine SSCs was performed in 0.2% (wt / v) Agarose-based 3D hydrogel. Colony diameters of porcine SSCs cultured for 6 days in 2D and 3D microenvironments were determined under a microscope. Compared to the 2D microenvironment, the 3D microenvironment showed a significant increase in the diameter of the collinies. All data represent the mean ± SEM of four independent experiments. N / D = not detected. * p < 0.05.
Figure 8 shows the morphology and AP (alkaline phosphatase) activity of porcine SSCs cultured in two-dimensional (2D) and three-dimensional (3D) microenvironments. Separated pig SSCs were cultured on culture plates coated with porcine testicular stromal cells (PTSCs) feeder (A) for 7 days, whereas 3D 4-arm (B) and 8-arm (C) polyethylene glycol (PEG) -hydrogel, respectively. The AP activity was then analyzed by AP staining. In conclusion, porcine colonies cultured in 3D 4-arm (E) and 8-arm (F) PEG-hydrogel displayed a thicker red and larger colony size than colonies grown on PTSCs feeder (D) This suggests that 3D 4-arm and 8-arm PEG-hydrogel can more effectively support the undifferentiated maintenance of porcine SSCs than the 2D microenvironment. The scale bar is 50 μm.
FIG. 9 shows the effect of 3D nisH using PEG-hydrogel for transcriptional regulation of self-regenerating and SSC-specific genes in porcine SSCs. After incubation for 7 days in neonatal male pig-derived SSCs in 2D PTSCs feeder phase and 3D 8-arm PEG-hydrogel, the transcription levels of self-renewal and SSC-specific genes were analyzed by real time PCR. As a result, 3D 8-arm PEG hydrogel pork SSCs cultured in 2D is PTSCs feeder than SSC- specific gene THY1 (D), and significantly higher expression of the UCHL1 (E) and the magnetic reproducing genes OCT4 (A ), NANOG (B) and EPCAM (C) were not detected in 2D and 3D microenvironments. The error bar indicates the standard error (SE). n = 5. * p < 0.05.
The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.
Example One; animal
Male piglets were donated at 1- to 4-day old hybrid (Landrace X Yorkshire or Yorkshire X Yorkshire) bonsai (Gumbo, Wonju, Korea) and the separation of the testicles from neonatal piglets was performed using conventional castration Lt; / RTI > All animal surgeries were performed in accordance with the IACUC approval No. KW-131106-1 and guidelines of the Ethical Commission for Animal Experimentation, Kangwon National University.
Example 2: Collection of spermatogonial stem cells from newborn pig testicles
The recovery of spermatogonial stem cells from the new born pig testicles was performed using a conventional DP (Petri dish plating post-differential plating) method. In summary, seminiferous tubules from testis were mechanically dispersed and enzymatically treated with type IV collagenase (Worthington Biochemical, Lakewood, Calif.). Hyaluronidase (Sigma-Aldrich, St. Louis, Mo.) and trypsin (Welgene Inc., Daegu, Korea) were used continuously and individually to disperse the cleaved tubules. Then, dispersed cells without erythrocytes were treated with 10% (v / v) fetal bovine serum (FBS; Welgene) and 1% (v / v) antibiotic-antimycotic The cells were cultured in gelatin-coated Petri dishes in high glucose Dulbecco's modified Eagle medium (DMEM; Welgene) supplemented with Welgene, and the supernatant cells were cultured for 16 hours. For the Petri dish plating post-DP, the collected cells were resuspended in 15% (v / v) FBS, 1% (v / v) nonessential amino acids (NEAA; Invitrogen, Carlsbad, Calif.), 1% -antimycotic solution, 2 mM L-glutamine (Invitrogen ), 0.1 mM beta -mercaptoethanol (Invitrogen), 10 3 units / ml mouse LIF (leukemia inhibitory factor; Chemicon International Inc., Temecula, CA), and 10 ng / ml GDNF (R & D Systems Inc., Minneapolis, MN) supplemented with glial cell-derived neurotrophic factor (DMEM).
After overnight, cells not attached to the bottom of the Petri dish were carefully withdrawn and the recovered cells used in the next experiment
Example 3: Construction of an agarose-based three-dimensional hydrogel and encapsulation of porcine SSCs in a hydrogel
Substrate stock solutions were prepared by dissolving 1% (w / v) Agarose powder in DMEM under heat conditions. Next, to construct an agarose-based 3D hydrogel with various mechanical properties, the stock was diluted to various concentrations (0.2 and 0.6% (w / v)) in DMEM heated to 37 ° C and encapsulated in a hydrogel of porcine SSCs It was solidified from a mixed cell and a substrate solution, and humidity of 95% at 31 ℃ air and 5% CO 2 conditions, diluted in 37 ℃.
Example 4: Culture of porcine SSCs
For 2D cultures, 4 x 10 < 5 > porcine SSCs were plated on Mitomycin C coated or uncoated culture plates with mitotically inactivated STO cells. At the same time, for 3D culture, 1 x 10 < 5 > porcine SSCs were inserted into an agarose-based three-dimensional hydrogel. Subsequently, pig SSCs exposed to 2-D and 3-D microenvironments were incubated with ITS (insulin-transferrin-selenium; Invitrogen), 60 μM putrescine dihydrochloride (Sigma-Aldrich), 6 mg / (Sigma-Aldrich), 5 mg / ml bovine serum albumin (BSA, Sigma-Aldrich), 2 mM L (Sigma-Aldrich), 1% (v / v) antibiotic-antimycotic solution, 0.1 mM glutamine, 0.05 mM beta-mercaptoethanol, 1% (v / v) FBS, 1% (Sigma-Aldrich), 10 3 units / ml mouse LIF, 10 ng / ml GDNF, 30 ng / ml beta-estradiol (Sigma-Aldrich), 60 ng / ml progesterone (Invitrogen) supplemented with human EGF (epidermal growth factor; Peprotech, Inc., Rocky Hill, NJ) and 10 ng / ml human bFGF Lt; / RTI > for 6 days. The medium was replaced with fresh every 2 days.
Example 5: Trippane blue exclusion assay
Ten microliters of 0.4% (wt / v) trypan blue solution (Sigma-Aldrich) were mixed with 10 μL cell suspension in culture medium and the number of positively stained cells was counted using a hemocytometer. Dead cells fail to exclude dye and are positively stained, and viable cells are dyed negatively by excluding the dye. To analyze cell viability, the percentage of viable cells was calculated based on the total number of cells (survival + death).
Example 6: AP (Alkaline Phosphatase) staining
Cells immobilized with 4% (v / v) paraformaldehyde (Junsei Chemical Co., Ltd., Chuo-ku, Japan) were washed with DPBS and incubated with 0.2 mg / ml Naphthol AS-MX phosphate (Sigma-Aldrich) (Sigma-Aldrich) supplemented with 0.1 M pH 8.2 Tris buffer (Sigma-Aldrich) supplemented with 1 mg / ml v / v N-dimethyl formamide Respectively. The stained cells were rinsed twice with DPBS and the percentage of AP-positive cells was measured under a microscope (CKX-41; Olympus, Tokyo, Japan) using a hemocytometer.
Example 7: Measurement of colony diameter
Colony diameters were calculated by averaging the horizontal and vertical diameters of the colonies measured using i-Solution Lite version 10.0 software (IMT i-Solution Inc., Vancouver, Canada).
Example 8 Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
Total mRNA was extracted using Dynabeads mRNA Direct ™ Kit (Ambion, Austin, Tex.), And cDNA synthesis was then performed using ReverTra Ace qPCR RT Master Mix using gDNA Remover (Toyobo, Osaka, Japan ). Next, qRT-PCR was performed on a 7500 Real time PCR system (Applied Biosystem, Foster City, CA) with THUNDERBIRD SYBR qPCR Mix (Toyobo). PCR specificity was analyzed by analyzing the melting curve data and normalization of specific gene expression was performed by comparing the mRNA level of GAPDH . The relative mRNA level was calculated as 2 - ΔΔ Ct , where Ct = threshold cycle for target amplification, ΔCt = Ct target gene (specific genes for each sample) - Ct internal reference GAPDH), and ΔΔCt = ΔCt sample (treated sample in each experiment) - ΔCt calibrator (control sample in each experiment). Table 1 shows the general information and sequences of primers designed by Primer3 software (Whitehead Institute / MIT Center for Genome Research) with cDNA sequences derived from GenBank against pigs.
Table 1 shows the primer sequences and PCR conditions used in the present invention. In Table 1, the reference 1 is Park MH1, Park JE, Kim MS, Lee KY, Park HJ, Yun JI, Choi JH, Lee ES, Lee ST. Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. J Assist Reprod Genet. 2014 Aug; 31 (8): 983-91. doi: 10.1007 / s10815-014-0271-7. Epub 2014 Jun 18.
Example 9: Fluorescence Immune assay
Immobilization was performed by incubating the cells in 4% (v / v) paraformaldehyde solution for 15 min. After immersing the immobilized cells in DPBS, the immobilized cells were transferred to DPBS supplemented with 0.01% (v / v) Triton X-100 (Sigma-Aldrich) and incubated with anti-rabbit OCT3 / 4, NANOG, SOX2, PLZF, TRA-1-60 and TRA-1-81 primary antibodies. Primary antibodies were then detected using an Alexa Flour 488-conjugated secondary antibody diluted in DPBS. Details of the primary or secondary antibody and dilution rates are listed in Table 2. After 1 hour, the stained cells were washed twice with DPBS, and the fluorescence intensity derived from them was measured using SoftMaxPro 6.2.2. (Molecular Devices Cooperation, Sunnyvale, Calif.).
Table 2 shows the primary and secondary antibodies used in the present invention and their related information. In Table 2, the reference 1 is Park MH1, Park JE, Kim MS, Lee KY, Park HJ, Yun JI, Choi JH, Lee ES, Lee ST. Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. J Assist Reprod Genet. 2014 Aug; 31 (8): 983-91. doi: 10.1007 / s10815-014-0271-7. Epub 2014 Jun 18.
Example 10: Statistical analysis
SAS (Statistical Analysis System) software was used to statistically analyze all numerical data from each experiment. When detecting the significance of the main effect through the ANOVA analysis in the SAS package, the comparison of each treatment was performed by least squares or DUNCAN method, and the significance difference in the treatments was determined when the p value was less than 0.05.
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| "ENGINEERING MATRIX PROPERTIES FOR STEM CELL CULTURE AND GENE DELIVERY ". Doctor of Philosophy in Chemical Engineering in the Graduate College of the University of Illinois at Urbana-Champaign, (2012.* |
| Asian J Androl. 17(6):972-80 (2015.12.)* |
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