Methods for Expansion of Human Embryonic Stem Cells
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《干细胞学杂志》
a Department of Obstetrics and Gynecology,
b Institute of Reproductive Medicine and Population, Medical Research Center, College of Medicine, Seoul National University, Korea;
c Central Research Institute, Sam Jin Pharm. Co., Ltd., Hwasung, Korea;
d R&D Center, Jeil Pharm. Co., Ltd., Yongin, Korea;
e Department of Physiology, Yonsei University College of Medicine, Seoul, Korea
Key Words. Human embryonic stem cells ? Stem cell expansion ? Mechanical transfer ? Enzymatic transfer ? Cell line maintenance
Correspondence: Shin Yong Moon, M.D., Ph.D., Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. Telephone: 82-2-2072-2384; Fax: 82-2-3672-7601; e-mail: shmoon@plaza.snu.ac.kr; and Dong-Wook Kim, Ph.D., Department of Physiology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. Telephone: 82-2-2228-1703; Fax: 82-2-393-0203; e-mail: dwkim2@yumc.yonsei.ac.kr
ABSTRACT
The first derivation of human embryonic stem cells (hESCs) from the inner cell mass of preimplanation blastocyst was reported in 1998 . Since then, several groups, including ours, have established new hESC lines . The derivation and characterization of hESCs have drawn much interest in respect to their potential use for direct cell therapy for human patients . However, unlike mouse ESC culture, manipulation of hESCs is a relatively delicate process and requires refined skills for expansion. Various techniques are used to expand established hESCs. Some groups mechanically transfer hESCs, whereas others use enzymes such as collagenase, trypsin, and dispase for expansion .
We introduce here methods of efficiently expanding our hESCs on STO feeder layers by both mechanical process and enzymatic treatment using collagenase IV. The selection of transfer method is based on experimental purpose. The mechanical transfer method requires a finely drawn Pasteur pipette to physically segregate the hESC colony into clumps of 150 to 200 cells for transfer. The advantages lie in the absence of cell-dissociating enzyme and the ability to isolate undifferentiated hESCs from differentiated cells. This process is ideal for maintaining hESC lines. However, mechanical transfers are laborious and time-consuming, making it difficult to process many cells at a time. The enzymatic transfer method, a faster and simpler method than the previous, uses the enzyme collagenase to separate hESCs from STO feeder layer. Once the colonies are isolated from the feeder layer by enzyme treatment followed by gentle pipetting, the colonies are pipetted into small cell clumps for transfer. However, these cell clumps vary in size, and in some cases, both differentiated and undifferentiated cells are transferred. This method is used to increase cell number for experiments that require large quantities of cells. The selection of transfer technique depends ultimately on the experimental purpose.
EXPANSION OF HESCs BY MECHANICAL TRANSFER
We used this method for experiments requiring large quantities of cells. A collagenase IV (Gibco-BRL/Invitrogen, Carlsbad, CA) solution was stored in 1-ml (2-mg/ml) aliquots at –70°C. The enzyme was thawed out at 37°C for 30–60 minutes before use.
The hESCs, grown on STO feeder layer in a 0.1% gelatin-coated 35-mm culture dish, were inspected before collagenase treatment, and at times differentiated hESCs were removed with a dissecting pipette. For transfer, hESC culture dish was washed with phosphate-buffered saline once and then treated with 1 ml of prewarmed collagenase at 37°C in 5% CO2 for approximately 30 minutes (Fig. 4A). The collagenase was removed, and 2 ml of culture medium was added as hESC colonies began to peel away from the surrounding STO feeder layer (Fig. 4B). The hESC colonies were gently pipetted with a 200-μl micropipette to detach from STO feeder layer (Figs. 4C, 4D). The isolated colonies were collected in a 15-ml conical tube with a 200-μl pipette, and medium was added to a final volume of 2 ml (Fig. 4E). The hESC colonies were allowed to settle to the bottom of the tube (~20 seconds). Once the hESC colonies settled, the supernatant containing single cell or STO cells was removed. This process was repeated twice before the colonies were made into small clumps by pipetting approximately five times with a 200-μl micropipette in a small volume of medium. The hESC clumps in 1-ml volume were spaced out evenly on a feeder layer of a 35-mm culture dish containing 1 ml of medium to a final volume of 2 ml (Fig. 4F). The dish was incubated at 37°C in 5% CO2 for 2 days to allow small clumps to attach to the dish. Two days after transfer of cells, the attachment of cells was verified under a stereomicroscope. Unattached cells were removed with a micropipette when 1 ml of old medium was replaced with 1 ml of new medium. The hESC morphology was inspected using phase-contrast microscope daily. The cells were cultured for approximately 5–7 days for transfer and were processed for experiments if cell quality and counts were sufficient after several passages.
Figure 4. Enzymatic transfer of human embryonic stem cells (hESCs) using collagenase IV. (A): Treatment of undifferentiated hESC colonies with collagenase. (B): After 30 minutes of enzyme treatment, the cells began to detach around the edges. At this time point, collagenase was removed and new medium was added. (C): The colonies lifted off the dish by gently pipetting with a 200-μl micropipette. (D): Multiple colonies completely detached from dish. (E): The detached hESC colonies were collected in a 15-ml conical tube, allowed to settle to bottom, and pipetted multiple times to make small clumps. (F): Small clumps transferred to new culture dish. Scale bar, 500 μm.
CONCLUSIONS
This research was supported by grants SC1020 and SC2140 from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. We thank Lim Andrew Lee for preparation of this manuscript.
REFERENCES
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Oh SK, Kim HS, Ahn HJ et al. Derivation and characterization of new human embryonic stem cell lines, SNUhES1, SNUhES2, and SNUhES3. STEM CELLS 2005;23:211–219.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Park JH, Kim SJ, Oh EJ et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 2003;69:2007–2014.
Cowan CA, Klimanskaya I, McMahon J etal. Derivation of embryonicstem cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.
Suss-Toby E, Gerecht-Nir S, Amit M et al. Derivation of a diploid human embryonic stem cell line from a mononuclear zygote. Hum Reprod 2004;19:670–675.
Heins N, Englund MC, Sjoblom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.
Hwang WS, Ryu YJ, Park JH et al. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 2004;303:1669–1674.
Lindvall O, Kokaia Z, Martinez-Serrano A. Stem cell therapy for human neurodegenerative disorders: how to make it work. Nat Med 2004;10: S42–S50.
Stojkovic M, Lako M, Strachan T et al. Derivation, growth and applications of human embryonic stem cells. Reproduction 2004;128:159–267.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells Hum Reprod 2003;18:1404–1409.
Inzunza J, Sahlen S, Holmberg K et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveal the occurrence of an isodicentric X chromosome after long-term cultivation. Mol Hum Reprod 2004;10:461–466.
Draper JS, Smith K, Gokhale P et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 2004;22:53–54.
Buzzard JJ, Gough NM, Crook JM et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004;22:381–382.
Mitalipova MM, Rao RR, Hoyer DM et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol 2005;23:19–20.(Sun Kyung Oha,b, Hee Sun )
b Institute of Reproductive Medicine and Population, Medical Research Center, College of Medicine, Seoul National University, Korea;
c Central Research Institute, Sam Jin Pharm. Co., Ltd., Hwasung, Korea;
d R&D Center, Jeil Pharm. Co., Ltd., Yongin, Korea;
e Department of Physiology, Yonsei University College of Medicine, Seoul, Korea
Key Words. Human embryonic stem cells ? Stem cell expansion ? Mechanical transfer ? Enzymatic transfer ? Cell line maintenance
Correspondence: Shin Yong Moon, M.D., Ph.D., Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. Telephone: 82-2-2072-2384; Fax: 82-2-3672-7601; e-mail: shmoon@plaza.snu.ac.kr; and Dong-Wook Kim, Ph.D., Department of Physiology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. Telephone: 82-2-2228-1703; Fax: 82-2-393-0203; e-mail: dwkim2@yumc.yonsei.ac.kr
ABSTRACT
The first derivation of human embryonic stem cells (hESCs) from the inner cell mass of preimplanation blastocyst was reported in 1998 . Since then, several groups, including ours, have established new hESC lines . The derivation and characterization of hESCs have drawn much interest in respect to their potential use for direct cell therapy for human patients . However, unlike mouse ESC culture, manipulation of hESCs is a relatively delicate process and requires refined skills for expansion. Various techniques are used to expand established hESCs. Some groups mechanically transfer hESCs, whereas others use enzymes such as collagenase, trypsin, and dispase for expansion .
We introduce here methods of efficiently expanding our hESCs on STO feeder layers by both mechanical process and enzymatic treatment using collagenase IV. The selection of transfer method is based on experimental purpose. The mechanical transfer method requires a finely drawn Pasteur pipette to physically segregate the hESC colony into clumps of 150 to 200 cells for transfer. The advantages lie in the absence of cell-dissociating enzyme and the ability to isolate undifferentiated hESCs from differentiated cells. This process is ideal for maintaining hESC lines. However, mechanical transfers are laborious and time-consuming, making it difficult to process many cells at a time. The enzymatic transfer method, a faster and simpler method than the previous, uses the enzyme collagenase to separate hESCs from STO feeder layer. Once the colonies are isolated from the feeder layer by enzyme treatment followed by gentle pipetting, the colonies are pipetted into small cell clumps for transfer. However, these cell clumps vary in size, and in some cases, both differentiated and undifferentiated cells are transferred. This method is used to increase cell number for experiments that require large quantities of cells. The selection of transfer technique depends ultimately on the experimental purpose.
EXPANSION OF HESCs BY MECHANICAL TRANSFER
We used this method for experiments requiring large quantities of cells. A collagenase IV (Gibco-BRL/Invitrogen, Carlsbad, CA) solution was stored in 1-ml (2-mg/ml) aliquots at –70°C. The enzyme was thawed out at 37°C for 30–60 minutes before use.
The hESCs, grown on STO feeder layer in a 0.1% gelatin-coated 35-mm culture dish, were inspected before collagenase treatment, and at times differentiated hESCs were removed with a dissecting pipette. For transfer, hESC culture dish was washed with phosphate-buffered saline once and then treated with 1 ml of prewarmed collagenase at 37°C in 5% CO2 for approximately 30 minutes (Fig. 4A). The collagenase was removed, and 2 ml of culture medium was added as hESC colonies began to peel away from the surrounding STO feeder layer (Fig. 4B). The hESC colonies were gently pipetted with a 200-μl micropipette to detach from STO feeder layer (Figs. 4C, 4D). The isolated colonies were collected in a 15-ml conical tube with a 200-μl pipette, and medium was added to a final volume of 2 ml (Fig. 4E). The hESC colonies were allowed to settle to the bottom of the tube (~20 seconds). Once the hESC colonies settled, the supernatant containing single cell or STO cells was removed. This process was repeated twice before the colonies were made into small clumps by pipetting approximately five times with a 200-μl micropipette in a small volume of medium. The hESC clumps in 1-ml volume were spaced out evenly on a feeder layer of a 35-mm culture dish containing 1 ml of medium to a final volume of 2 ml (Fig. 4F). The dish was incubated at 37°C in 5% CO2 for 2 days to allow small clumps to attach to the dish. Two days after transfer of cells, the attachment of cells was verified under a stereomicroscope. Unattached cells were removed with a micropipette when 1 ml of old medium was replaced with 1 ml of new medium. The hESC morphology was inspected using phase-contrast microscope daily. The cells were cultured for approximately 5–7 days for transfer and were processed for experiments if cell quality and counts were sufficient after several passages.
Figure 4. Enzymatic transfer of human embryonic stem cells (hESCs) using collagenase IV. (A): Treatment of undifferentiated hESC colonies with collagenase. (B): After 30 minutes of enzyme treatment, the cells began to detach around the edges. At this time point, collagenase was removed and new medium was added. (C): The colonies lifted off the dish by gently pipetting with a 200-μl micropipette. (D): Multiple colonies completely detached from dish. (E): The detached hESC colonies were collected in a 15-ml conical tube, allowed to settle to bottom, and pipetted multiple times to make small clumps. (F): Small clumps transferred to new culture dish. Scale bar, 500 μm.
CONCLUSIONS
This research was supported by grants SC1020 and SC2140 from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. We thank Lim Andrew Lee for preparation of this manuscript.
REFERENCES
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Oh SK, Kim HS, Ahn HJ et al. Derivation and characterization of new human embryonic stem cell lines, SNUhES1, SNUhES2, and SNUhES3. STEM CELLS 2005;23:211–219.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Park JH, Kim SJ, Oh EJ et al. Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 2003;69:2007–2014.
Cowan CA, Klimanskaya I, McMahon J etal. Derivation of embryonicstem cell lines from human blastocysts. N Engl J Med 2004;350:1353–1356.
Suss-Toby E, Gerecht-Nir S, Amit M et al. Derivation of a diploid human embryonic stem cell line from a mononuclear zygote. Hum Reprod 2004;19:670–675.
Heins N, Englund MC, Sjoblom C et al. Derivation, characterization, and differentiation of human embryonic stem cells. STEM CELLS 2004;22:367–376.
Hwang WS, Ryu YJ, Park JH et al. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 2004;303:1669–1674.
Lindvall O, Kokaia Z, Martinez-Serrano A. Stem cell therapy for human neurodegenerative disorders: how to make it work. Nat Med 2004;10: S42–S50.
Stojkovic M, Lako M, Strachan T et al. Derivation, growth and applications of human embryonic stem cells. Reproduction 2004;128:159–267.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001;19:971–974.
Richards M, Fong CY, Chan WK et al. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002;20:933–936.
Hovatta O, Mikkola M, Gertow K et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells Hum Reprod 2003;18:1404–1409.
Inzunza J, Sahlen S, Holmberg K et al. Comparative genomic hybridization and karyotyping of human embryonic stem cells reveal the occurrence of an isodicentric X chromosome after long-term cultivation. Mol Hum Reprod 2004;10:461–466.
Draper JS, Smith K, Gokhale P et al. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 2004;22:53–54.
Buzzard JJ, Gough NM, Crook JM et al. Karyotype of human ES cells during extended culture. Nat Biotechnol 2004;22:381–382.
Mitalipova MM, Rao RR, Hoyer DM et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol 2005;23:19–20.(Sun Kyung Oha,b, Hee Sun )