Effects of Type IV Collagen and Laminin on the Cryopreservation of Human Embryonic Stem Cells
http://www.100md.com
《干细胞学杂志》
a Division of Stem Cell Biology, Medical Research Center, MizMedi Hospital;
b Department of Life Science, College of Natural Sciences, Hanyang University;
c Stem Cell Research Center, Seoul, Korea
Key Words. Human embryonic stem cells ? Cryopreservation ? Type IV collagen ? Laminin
Correspondence: Hyun Soo Yoon, Ph.D., Division of Stem Cell Biology, Medical Research Center, MizMedi Hospital, 701-4 Naebalsan-dong, Kangseo-ku, Seoul 157–280, Korea. Telephone: 82-2-2007-1840; Fax: 82-2-2007-1852; e-mail: yoon@mizmedi.net
ABSTRACT
Human embryonic stem cells (hESCs) derived from the inner cell masses (ICMs) of blastocysts are pluripotent cells, which have the capacity to self-renew and to differentiate into a wide variety of tissues exhibiting characteristics of all three germ layers in vitro and in vivo and yet still retain a normal karyotype .
To exploit the remarkable potentials of hESCs, technical improvements for the handling, manipulation, and cryopreservation are an important part of hESC technology. The ability for successful cryopreservation of hES cells with limited loss in viability is essential for the success of widespread use of stem cells. Also, an effective freezing technique would allow the efficient preservation of stocks of early-passage hESCs, as well as the cryopreservation of specific clones developed from the original hESC lines such as genetically modified clones . However, optimal cryopreservation conditions of hESCs have not been established clearly because of high rates of cell death from dissociation of clumps and spontaneous differentiation of hESCs after thawing.
Because ES cells originate from the pluripotent cells of blastocysts and retain ES cell properties in culture , it has been postulated that a blastocyst cryopreservation method might be effective with ES cells . Recently, vitrification of hESCs by the open-pulled-straw technique has been reported as effective for their cryopreservation; all vitrified hESCs were recovered after thawing and retained the key properties of pluripotent stem cells. However, the vitrification of hESCs could increase the levels of cell death and spontaneous differentiation after thawing and limit the number of hESC clumps that can be cryopreserved simultaneously . Furthermore, a potential hazard of vitrification is the transmission of infective agents into cells .
The conventional slow freezing and rapid thawing method is most commonly used for the cryopreservation of embryos and cell lines. Although this standard method is efficient for the cryopreservation of mouse ES (mES) cells, the survival of undifferentiated hESCs after conventional slow freezing is very poor, with most cells either differentiating or dying . During slow freezing, there are many significant stresses on cells that can contribute to the loss of pluripotency, including osmotic stress, stresses to cell-junction and cell-transport systems, and disruption of organelles .
In particular, previous reports have indicated that the soluble factors and extracellular matrix (ECM) produced by feeder cells might be important for proliferation and maintenance of undifferentiated hESCs . ECM provides a platform for multiple signaling mechanisms, which may explain its importance in cell differentiation, migration, and survival . The basement membrane (BM) contains many molecules, including structural proteins, type IV collagen and laminin isotypes, and various glycoproteins rich in heparan sulfates . Regulation of their assembly plays a crucial role during development, and type IV collagen and laminin are essential for this process . In mES cells, the expression and accumulation of laminin and its companion matrix components on the cell surface and their assembly into the BM affect the differentiation of primitive endodermal and epiblast cells .
In this study, we evaluated the effects of type IV collagen and laminin in the freezing medium on the proliferation and differentiation of hESCs after slow freezing and rapid thawing.
MATERIALS AND METHODS
Effects of Type IV Collagen or Laminin Concentration on the Survival and Differentiation Rate of hESCs after Slow Freezing and Rapid Thawing
When frozen hESC clumps were recovered after conventional slow freezing and rapid thawing, most hESC clumps were not attached and formed undersized colonies to end up with differentiation. Compared with fresh controls (4–5 days), the cryopreserved group required prolonged cultures (7 days) to allow proliferation of undifferentiated cells. Also, morphological appearance of the colonies was improved with extended culture of cryopreserved hESCs (Figs. 1A–1D).
Figure 1. Morphological features and FACS analysis of hESCs after slow freezing and rapid thawing. Representative morphologies of hESCs grown on primary mouse embryonic fibroblasts were presented from colonies of fresh control (A), frozen control (B), type IV collagen treatment (C), and laminin-treated frozen hESCs (D) at day 7 after thawing. At high magnification, the undifferentiated hESCs exhibit distinct cell borders, a high nucleus-to-cytoplasm ratio, and prominent nucleoli. The type IV collagen–treated or laminin-treated groups contained compact undifferentiated colonies, as did the culture of fresh control cells, although the frozen control cells did not contain as many undifferentiated colonies as type IV collagen–treated or laminin-treated frozen groups. FACS analysis of fresh control (E), frozen control (F), type IV collagen–treated (G), and laminin-treated (H) frozen hESCs after slow freezing and thawing at third passage. Nonviable cells were identified using propidium iodide staining and excluded for the analysis. The horizontal marker in each plot is positioned to represent the fluorescence of ~ 99% of the appropriate fluorescein isotype–matched control; the vertical marker is positioned to represent the TRA-1-60–positive population. Values in each quadrant indicate the percentage of TRA-1-60–positive cells. Scale bars indicate 100 μm (A–D) and 10 μm (insets). Abbreviations: FACS, fluorescence-activated cell sorter; hESC, human embryonic stem cell.
To determine the effective concentration of type IV collagen and laminin treatments, we treated the type IV collagen or laminin at 1, 2, and 5 μg/ml in the freezing medium. The nonattached clumps after thawing were excluded to get the survival or differentiation rate after thawing. Compared with the frozen control group, the addition of type IV collagen (1 μg/ml) to the freezing medium significantly increased the survival rate (24 of 110 versus 40 of 110 ; p < .01) (Table 1). The increased concentration of type IV collagen (5 μg/ml) or laminin (5 μg/ml) did not improve the survival rate of hESCs. However, the differentiation rate was significantly reduced in the presence of type IV collagen or laminin. The addition of type IV collagen (1, 2, or 5 μg/ml) or laminin (1 or 2 μg/ml) to the freezing medium also significantly reduced the differentiation rate (p < .01) (Table 1). The addition of ECMs in the thawing medium had no effect on the survival and differentiation rates (data not shown). Therefore, the addition of either 1 μg/ml type IV collagen or 1 μg/ml laminin only to the freezing medium was selected in subsequent experiments.
Table 1. Effects of ECM, type IV collagen, and laminin on the survival and differentiation rates of hES cells after slow freezing and rapid thawing
The addition of type IV collagen (1 μg/ml) to the freezing medium significantly increased the survival rate (60 of 249 versus 96 of 262 ; p < .01). At third passage after thawing, the rate of spontaneous differentiation was lower in type IV collagen–treated or laminin-treated frozen hES colonies than that of frozen control colonies (35 of 332 and 23 of 202 versus 89 of 254 ; p < .01), and these values were similar to those of a fresh control group subcultured without cryopreservation (20 of 245 ) (Table 2). Addition of type IV collagen or laminin to freezing medium resulted in the decreased differentiation rate of hESCs after slow freezing.
Table 2. Survival and differentiation rates of hES cells after slow freezing and rapid thawing using the freezing medium containing either type IV collagen (1 μg/ml) or laminin (1 μg/ml)
FACS Analysis of Frozen-Thawed hESCs
To analyze the undifferentiation state of hESCs at the third passage after thawing, we compared the expression of TRA-1-60 in the fresh control, frozen control, type IV collagen–treated, and laminin-treated hESCs by FACS analysis. TRA-1-60 expression in hESCs was in the range of 93%–95% in fresh control (n = 5 to 13 in three cell lines), 90%–93% in type IV collagen–treated cells (n = 5 to 11 in three cell lines), 88%–93% in laminin-treated cells (n = 5 to 11 in three cell lines), and 68%–72% in frozen control cells (n = 5 to 11 in three cell lines), respectively (Figs. 1E–1H). TRA-1-60 expression was statistically significant among the fresh control group (94.2 ± 1.2%), type IV collagen–treated group (91.7 ± 1.5%), laminin-treated group (90.4 ± 1.1%), and frozen control group (69.7 ± 1.6%) (Student’s t-test; fresh control versus frozen control, p < .01; type IV collagen–treated or laminin-treated versus frozen control groups, p < .01). Addition of type IV collagen or laminin increased the undifferentiation status of hESCs after thawing.
Effect of Type IV Collagen or Laminin Treatment on the Transcription of Genes Encoding Undifferentiated hESC Markers
We investigated the expression of transcription factors known to be associated with pluripotency state. Several transcription factors expressed in undifferentiated mES cells and hESCs with decreased expression on differentiation were evaluated in the fresh control group, frozen control group, and type IV collagen–treated or laminin-treated hESCs using quantitative RT-PCR. Significant difference was observed between the frozen control group and type IV collagen–treated or laminin-treated hESCs (Table 3). Oct-4 and Rex-1 expression was abundant in the fresh control group, type IV collagen–treated hESCs (0.822, 0.809), and laminin-treated hESCs (0.816, 0.801) but was significantly decreased in the frozen control hESCs (0.212, 0.234). In this result, Oct-4 and Rex-1 expression was restored by addition of type IV collagen or laminin treatment to freezing medium.
Table 3. Results of real-time polymerase chain reaction for undifferentiated human embryonic stem cell marker expression after treatment with type IV collagen or laminin
Rescue of Basement Membrane-Related Gene Expression by Type IV Collagen or Laminin Treatment during Slow Freezing and Rapid Thawing
To analyze the effect of type IV collagen and laminin on BM of hESCs during cryopreservation, the steady-state transcription level of specific BM components was examined by semiquantitative RT-PCR. The addition of type IV collagen (1 μg/ml) or laminin (1 μg/ml) to the freezing medium affected the expression level of the BM-related gene of hESCs after thawing (Fig. 2A). The expression of collagen type IV 1, laminin 1, fibronectin 1, nidogen, and perlecan was significantly increased in type IV collagen–treated or laminin-treated groups compared with the frozen control group (p < .05). The BM-related gene expression in type IV collagen–treated or laminin-treated groups was similar to that of the fresh control group (Figs. 2B–2G). Our results clearly show that type IV collagen or laminin treatment could rescue BM-related gene expression.
Figure 2. Effects of type IV collagen and laminin treatment on the expression of genes encoding BM proteins after hES cryopreservation. (A): Expression of various BM-related genes in hESCs. Total RNA was extracted from fresh and frozen control cells, as well as from type IV collagen–treated or laminin-treated hESCs after thawing. Lane 1: fresh control;, Lane 2: frozen control; Lane 3: type IV collagen treated; Lane 4: laminin treated. Various BM gene expression profiles were changed. Semiquantitative reverse transcription–polymerase chain reaction of the following BM proteins after thawing was performed: collagen type IV 1 (B), dystroglycan 1 (C), fibronectin 1 (D), laminin 1 (E), nidogen (F), and perlecan (G). Expression levels of BM-related genes were normalized to b-actin expression levels and are shown as relative quantification. Data are shown as mean ± standard deviation (n = 3). *p <.05, frozen control group versus type IV collagen–treated or laminin-treated group. Abbreviations: BM, basement membrane; Coll, type IV collagen (1 μg/ml); hESC, human embryonic stem cell; Lami, laminin (1 μg/ml).
Characterization of hESCs after Slow Freezing and Rapid Thawing
The addition of type IV collagen or laminin to the freezing medium could maintain the key properties of Miz-hES1 cells after slow freezing and rapid thawing. hESCs were analyzed immunocytochemically using a series of antibodies against cell-surface carbohydrate antigens after thawing, which are specifically expressed on undifferentiated hESCs. They exhibited high levels of alkaline phosphatase (AP) activity (Fig. 3A). SSEA-1 was not expressed in undifferentiated hESCs (Fig. 3B). SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 were strongly expressed (Figs. 3C–3F). Also, Oct-4 was expressed specifically in undifferentiated hESCs but not in differentiated cells (Figs. 4A, 4B). When karyotype analysis was examined at passage 30 after thawing, hESCs had a normal 46 XY karyotype (Fig. 4C). Similar results were obtained from two other cell lines (Miz-hES2 and Miz-hES3) (data not shown).
Figure 3. Marker expression in Miz-hES1 cells after addition of type IV collagen or laminin in freezing medium. Staining of Miz-hES1 colonies with alkaline phosphatase (A), SSEA-1 (B), SSEA-3 (C), SSEA-4 (D), TRA-1-60 (E), and TRA-1-81 (F). Colonies consisting of stained, central, and undifferentiated cells were strongly stained by antibodies of each surface marker protein. Scale bars indicate 100 μm.
Figure 4. Characterization of type IV collagen–treated or laminin-treated Miz-hES1 cell line at passage 30 after thawing. (A): Immunostaining of Miz-hES1 cells with anti–Oct-4 antibody. Oct-4 was expressed homogeneously in undifferentiated hESCs. (B): Oct-4 expression by RT-PCR. Oct-4 was expressed only in undifferentiated Miz-hES1 cells (not in differentiated Miz-hES1 cells). Lane 1: 100-bp DNA ladder; lane 2: negative control; lane 3: primary mouse embryonic fibroblasts; lane 4: undifferentiated Miz-hES1 cells; lane 5: differentiated Miz-hES1 cells (day 30). The sizes of PCR product of the human ?-actin, mouse b-actin, and human Oct-4 primers are 838, 540, and 445 bp, respectively. (C): Karyotype analysis of hESC lines using G-band method. The karyotype of Miz-hES1 was examined at passage 30 after thawing. The karyotype was found to be normal (46 XY). Abbreviations: hESC, human embryonic stem cell; RT-PCR, reverse transcription–polymerase chain reaction.
Differentiation of Frozen and Thawed hESCs In Vivo
The developmental potential of the addition of type IV collagen or laminin on frozen-thawed hESCs was examined in vivo using a teratoma model. The three hESC lines (Miz-hES1, Miz-hES2, and Miz-hES3 of 44, 51, and 36 passages after thawing, respectively) used in this research formed teratomas after injection into SCID mice. Each teratoma contained representative tissues of the three embryonic germ layers, including endoderm (glandular-like tissue), mesoderm (osteoid-like tissue, cartilage, and adipocyte), and ectoderm (neural tube components). Representative tissues of three germ layers from frozen and thawed hESCs are demonstrated (Fig. 5).
Figure 5. In vivo differentiation of type IV collagen–treated or laminin-treated frozen-thawed human embryonic stem cells. (A): Choroids plexus-like tissue from frozen-thawed Miz-hES1 cells. (B): Cartilage-like tissue from frozen-thawed Miz-hES1 cells. (C): Osteoid- and marrow-like tissue from frozen-thawed Miz-hES2 cells. (D): Neural tube-like tissue from frozen-thawed Miz-hES3 cells. (E):Adipocyte-like cells from frozen-thawed Miz-hES2 cells. (F): Glandular-like tissue from frozen-thawed Miz-hES3 cells. Magnification, x100. Scale bars indicate 100 μm.
DISCUSSION
We thank Dr. Douglas N. Foster at the University of Minnesota and Dr. Seungkwon You at Korea University for proofreading the manuscript. This research was supported by grants M102KL010001-02K1201-00310 and M102KL010001-03K1201-00610 from the Stem Cell Research Center of the 21st Century Frontier Research Program, funded by the Ministry of Science and Technology, Republic of Korea.
REFERENCES
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.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;8:399–404.
Gearheart J. New Potential for human embryonic stem cells. Science 1998;282:1061–1062.
Bongso A, Fong CY, Ng SC et al. Isolation and culture of inner cell mass cells from human blastocysts. Hum Reprod 1994;9:2110–2117.
Freshney RI. Culture of Animal Cells: A Manual of Basic Technique. New York: Wiley-Liss Inc., 1994:255–265.
Reubinoff BE, Pera MF,Vajta G et al. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001;16:2187–2194.
Hawkins AE, Zuckerman MA, Briggs M et al. Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank. J Virol Methods 1996;60:81–88.
Tedder RS, Zuckerman MA, Goldstone AH et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995;46:137–140.
Frederickx V, Michiels A, Goossens E et al. Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells. Hum Reprod 2004;19:948–953.
Woods EJ, Liu J, Pollok K et al. A theoretically optimized method for cord blood stem cell cryopreservation. J Hematother Stem Cell Res 2003;12:341–50.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotech 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. Nature Biotech 2002; 20:933–936.
Li X, Chen Y, Scheele S et al. Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body. J Cell Biol 2001;153:811–822.
Kleinman HK, McGarvey ML, Liotta LA et al. Isolation and characterization of Type IV procollagen, laminin, and heparan sulfate proteglycan from the EHS sarcoma. Biochemistry 1982;21:6188–6193.
Ekblom P,Vestweber D, Kemler R. Cell-matrix interactions and cell adhesion during development. Annu Rev Cell Biol 1986;2:27–47.
Li S, Harrison D, Carbonetto S et al. Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation. J Cell Biol 2002;157:1279–1290.
Jaroslaw C, Anna W. Embryonic stem cell differentiation: The role of extracellular factors. Differentiation 2001;68:167–174.
Cooper S, Pera MF, Bennett W et al. A novel keratan sulfate proteoglycan from a human embryonal carcinoma cell-line. Biochem J 1992;286:959–966.
Carpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 2004;229:243–258.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation of self-renewal of ES cells. Nat Genet 2000;24:372–376.
Andrews PW, Oosterhuis JW, Damjanov I. Cell lines from human germ cell lines. In: Robertson EJ, ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford, U.K.: IRL Press, 1987:207–24.
Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic feeder layers which support the growth of human embryonic stem cells. Proteomics 2002;2:1187–1203.
Zhu X, Assoian K. Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell 1995;6:273–282.
Srebrow A, Friedmann Y, Ravanpay A et al. Expression of Hoxa-1 and Hoxb-7 is regulated by extracellular matrix-dependent signals in mammary epithelial cells. J Cell Biochem 1997;69:377–391.
Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 2002;12:432–438.
Cooper M, Tamura N, Quaranta V. The major laminin receptor of mouse embryonic stem cells is a novel isoform of the a6b1 integrin. J Cell Biol 1991;115:843–850.
XIaofeng L, Ulrika T, Jan T et al. Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane. Proc Natl Acad Sci U S A 2001;98:14416–14421.
Chen Y, Li X, Eswarakumar P et al. Fibroblast growth factor (FGF) signaling through PI3-kinase and Akt/PKB is required for embryoid body differentiation. Oncogene 2000;19:3750–3756.
Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science 2000;287:1427–1430.
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.(Sun Jong Kima,b, Jong Hyu)
b Department of Life Science, College of Natural Sciences, Hanyang University;
c Stem Cell Research Center, Seoul, Korea
Key Words. Human embryonic stem cells ? Cryopreservation ? Type IV collagen ? Laminin
Correspondence: Hyun Soo Yoon, Ph.D., Division of Stem Cell Biology, Medical Research Center, MizMedi Hospital, 701-4 Naebalsan-dong, Kangseo-ku, Seoul 157–280, Korea. Telephone: 82-2-2007-1840; Fax: 82-2-2007-1852; e-mail: yoon@mizmedi.net
ABSTRACT
Human embryonic stem cells (hESCs) derived from the inner cell masses (ICMs) of blastocysts are pluripotent cells, which have the capacity to self-renew and to differentiate into a wide variety of tissues exhibiting characteristics of all three germ layers in vitro and in vivo and yet still retain a normal karyotype .
To exploit the remarkable potentials of hESCs, technical improvements for the handling, manipulation, and cryopreservation are an important part of hESC technology. The ability for successful cryopreservation of hES cells with limited loss in viability is essential for the success of widespread use of stem cells. Also, an effective freezing technique would allow the efficient preservation of stocks of early-passage hESCs, as well as the cryopreservation of specific clones developed from the original hESC lines such as genetically modified clones . However, optimal cryopreservation conditions of hESCs have not been established clearly because of high rates of cell death from dissociation of clumps and spontaneous differentiation of hESCs after thawing.
Because ES cells originate from the pluripotent cells of blastocysts and retain ES cell properties in culture , it has been postulated that a blastocyst cryopreservation method might be effective with ES cells . Recently, vitrification of hESCs by the open-pulled-straw technique has been reported as effective for their cryopreservation; all vitrified hESCs were recovered after thawing and retained the key properties of pluripotent stem cells. However, the vitrification of hESCs could increase the levels of cell death and spontaneous differentiation after thawing and limit the number of hESC clumps that can be cryopreserved simultaneously . Furthermore, a potential hazard of vitrification is the transmission of infective agents into cells .
The conventional slow freezing and rapid thawing method is most commonly used for the cryopreservation of embryos and cell lines. Although this standard method is efficient for the cryopreservation of mouse ES (mES) cells, the survival of undifferentiated hESCs after conventional slow freezing is very poor, with most cells either differentiating or dying . During slow freezing, there are many significant stresses on cells that can contribute to the loss of pluripotency, including osmotic stress, stresses to cell-junction and cell-transport systems, and disruption of organelles .
In particular, previous reports have indicated that the soluble factors and extracellular matrix (ECM) produced by feeder cells might be important for proliferation and maintenance of undifferentiated hESCs . ECM provides a platform for multiple signaling mechanisms, which may explain its importance in cell differentiation, migration, and survival . The basement membrane (BM) contains many molecules, including structural proteins, type IV collagen and laminin isotypes, and various glycoproteins rich in heparan sulfates . Regulation of their assembly plays a crucial role during development, and type IV collagen and laminin are essential for this process . In mES cells, the expression and accumulation of laminin and its companion matrix components on the cell surface and their assembly into the BM affect the differentiation of primitive endodermal and epiblast cells .
In this study, we evaluated the effects of type IV collagen and laminin in the freezing medium on the proliferation and differentiation of hESCs after slow freezing and rapid thawing.
MATERIALS AND METHODS
Effects of Type IV Collagen or Laminin Concentration on the Survival and Differentiation Rate of hESCs after Slow Freezing and Rapid Thawing
When frozen hESC clumps were recovered after conventional slow freezing and rapid thawing, most hESC clumps were not attached and formed undersized colonies to end up with differentiation. Compared with fresh controls (4–5 days), the cryopreserved group required prolonged cultures (7 days) to allow proliferation of undifferentiated cells. Also, morphological appearance of the colonies was improved with extended culture of cryopreserved hESCs (Figs. 1A–1D).
Figure 1. Morphological features and FACS analysis of hESCs after slow freezing and rapid thawing. Representative morphologies of hESCs grown on primary mouse embryonic fibroblasts were presented from colonies of fresh control (A), frozen control (B), type IV collagen treatment (C), and laminin-treated frozen hESCs (D) at day 7 after thawing. At high magnification, the undifferentiated hESCs exhibit distinct cell borders, a high nucleus-to-cytoplasm ratio, and prominent nucleoli. The type IV collagen–treated or laminin-treated groups contained compact undifferentiated colonies, as did the culture of fresh control cells, although the frozen control cells did not contain as many undifferentiated colonies as type IV collagen–treated or laminin-treated frozen groups. FACS analysis of fresh control (E), frozen control (F), type IV collagen–treated (G), and laminin-treated (H) frozen hESCs after slow freezing and thawing at third passage. Nonviable cells were identified using propidium iodide staining and excluded for the analysis. The horizontal marker in each plot is positioned to represent the fluorescence of ~ 99% of the appropriate fluorescein isotype–matched control; the vertical marker is positioned to represent the TRA-1-60–positive population. Values in each quadrant indicate the percentage of TRA-1-60–positive cells. Scale bars indicate 100 μm (A–D) and 10 μm (insets). Abbreviations: FACS, fluorescence-activated cell sorter; hESC, human embryonic stem cell.
To determine the effective concentration of type IV collagen and laminin treatments, we treated the type IV collagen or laminin at 1, 2, and 5 μg/ml in the freezing medium. The nonattached clumps after thawing were excluded to get the survival or differentiation rate after thawing. Compared with the frozen control group, the addition of type IV collagen (1 μg/ml) to the freezing medium significantly increased the survival rate (24 of 110 versus 40 of 110 ; p < .01) (Table 1). The increased concentration of type IV collagen (5 μg/ml) or laminin (5 μg/ml) did not improve the survival rate of hESCs. However, the differentiation rate was significantly reduced in the presence of type IV collagen or laminin. The addition of type IV collagen (1, 2, or 5 μg/ml) or laminin (1 or 2 μg/ml) to the freezing medium also significantly reduced the differentiation rate (p < .01) (Table 1). The addition of ECMs in the thawing medium had no effect on the survival and differentiation rates (data not shown). Therefore, the addition of either 1 μg/ml type IV collagen or 1 μg/ml laminin only to the freezing medium was selected in subsequent experiments.
Table 1. Effects of ECM, type IV collagen, and laminin on the survival and differentiation rates of hES cells after slow freezing and rapid thawing
The addition of type IV collagen (1 μg/ml) to the freezing medium significantly increased the survival rate (60 of 249 versus 96 of 262 ; p < .01). At third passage after thawing, the rate of spontaneous differentiation was lower in type IV collagen–treated or laminin-treated frozen hES colonies than that of frozen control colonies (35 of 332 and 23 of 202 versus 89 of 254 ; p < .01), and these values were similar to those of a fresh control group subcultured without cryopreservation (20 of 245 ) (Table 2). Addition of type IV collagen or laminin to freezing medium resulted in the decreased differentiation rate of hESCs after slow freezing.
Table 2. Survival and differentiation rates of hES cells after slow freezing and rapid thawing using the freezing medium containing either type IV collagen (1 μg/ml) or laminin (1 μg/ml)
FACS Analysis of Frozen-Thawed hESCs
To analyze the undifferentiation state of hESCs at the third passage after thawing, we compared the expression of TRA-1-60 in the fresh control, frozen control, type IV collagen–treated, and laminin-treated hESCs by FACS analysis. TRA-1-60 expression in hESCs was in the range of 93%–95% in fresh control (n = 5 to 13 in three cell lines), 90%–93% in type IV collagen–treated cells (n = 5 to 11 in three cell lines), 88%–93% in laminin-treated cells (n = 5 to 11 in three cell lines), and 68%–72% in frozen control cells (n = 5 to 11 in three cell lines), respectively (Figs. 1E–1H). TRA-1-60 expression was statistically significant among the fresh control group (94.2 ± 1.2%), type IV collagen–treated group (91.7 ± 1.5%), laminin-treated group (90.4 ± 1.1%), and frozen control group (69.7 ± 1.6%) (Student’s t-test; fresh control versus frozen control, p < .01; type IV collagen–treated or laminin-treated versus frozen control groups, p < .01). Addition of type IV collagen or laminin increased the undifferentiation status of hESCs after thawing.
Effect of Type IV Collagen or Laminin Treatment on the Transcription of Genes Encoding Undifferentiated hESC Markers
We investigated the expression of transcription factors known to be associated with pluripotency state. Several transcription factors expressed in undifferentiated mES cells and hESCs with decreased expression on differentiation were evaluated in the fresh control group, frozen control group, and type IV collagen–treated or laminin-treated hESCs using quantitative RT-PCR. Significant difference was observed between the frozen control group and type IV collagen–treated or laminin-treated hESCs (Table 3). Oct-4 and Rex-1 expression was abundant in the fresh control group, type IV collagen–treated hESCs (0.822, 0.809), and laminin-treated hESCs (0.816, 0.801) but was significantly decreased in the frozen control hESCs (0.212, 0.234). In this result, Oct-4 and Rex-1 expression was restored by addition of type IV collagen or laminin treatment to freezing medium.
Table 3. Results of real-time polymerase chain reaction for undifferentiated human embryonic stem cell marker expression after treatment with type IV collagen or laminin
Rescue of Basement Membrane-Related Gene Expression by Type IV Collagen or Laminin Treatment during Slow Freezing and Rapid Thawing
To analyze the effect of type IV collagen and laminin on BM of hESCs during cryopreservation, the steady-state transcription level of specific BM components was examined by semiquantitative RT-PCR. The addition of type IV collagen (1 μg/ml) or laminin (1 μg/ml) to the freezing medium affected the expression level of the BM-related gene of hESCs after thawing (Fig. 2A). The expression of collagen type IV 1, laminin 1, fibronectin 1, nidogen, and perlecan was significantly increased in type IV collagen–treated or laminin-treated groups compared with the frozen control group (p < .05). The BM-related gene expression in type IV collagen–treated or laminin-treated groups was similar to that of the fresh control group (Figs. 2B–2G). Our results clearly show that type IV collagen or laminin treatment could rescue BM-related gene expression.
Figure 2. Effects of type IV collagen and laminin treatment on the expression of genes encoding BM proteins after hES cryopreservation. (A): Expression of various BM-related genes in hESCs. Total RNA was extracted from fresh and frozen control cells, as well as from type IV collagen–treated or laminin-treated hESCs after thawing. Lane 1: fresh control;, Lane 2: frozen control; Lane 3: type IV collagen treated; Lane 4: laminin treated. Various BM gene expression profiles were changed. Semiquantitative reverse transcription–polymerase chain reaction of the following BM proteins after thawing was performed: collagen type IV 1 (B), dystroglycan 1 (C), fibronectin 1 (D), laminin 1 (E), nidogen (F), and perlecan (G). Expression levels of BM-related genes were normalized to b-actin expression levels and are shown as relative quantification. Data are shown as mean ± standard deviation (n = 3). *p <.05, frozen control group versus type IV collagen–treated or laminin-treated group. Abbreviations: BM, basement membrane; Coll, type IV collagen (1 μg/ml); hESC, human embryonic stem cell; Lami, laminin (1 μg/ml).
Characterization of hESCs after Slow Freezing and Rapid Thawing
The addition of type IV collagen or laminin to the freezing medium could maintain the key properties of Miz-hES1 cells after slow freezing and rapid thawing. hESCs were analyzed immunocytochemically using a series of antibodies against cell-surface carbohydrate antigens after thawing, which are specifically expressed on undifferentiated hESCs. They exhibited high levels of alkaline phosphatase (AP) activity (Fig. 3A). SSEA-1 was not expressed in undifferentiated hESCs (Fig. 3B). SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 were strongly expressed (Figs. 3C–3F). Also, Oct-4 was expressed specifically in undifferentiated hESCs but not in differentiated cells (Figs. 4A, 4B). When karyotype analysis was examined at passage 30 after thawing, hESCs had a normal 46 XY karyotype (Fig. 4C). Similar results were obtained from two other cell lines (Miz-hES2 and Miz-hES3) (data not shown).
Figure 3. Marker expression in Miz-hES1 cells after addition of type IV collagen or laminin in freezing medium. Staining of Miz-hES1 colonies with alkaline phosphatase (A), SSEA-1 (B), SSEA-3 (C), SSEA-4 (D), TRA-1-60 (E), and TRA-1-81 (F). Colonies consisting of stained, central, and undifferentiated cells were strongly stained by antibodies of each surface marker protein. Scale bars indicate 100 μm.
Figure 4. Characterization of type IV collagen–treated or laminin-treated Miz-hES1 cell line at passage 30 after thawing. (A): Immunostaining of Miz-hES1 cells with anti–Oct-4 antibody. Oct-4 was expressed homogeneously in undifferentiated hESCs. (B): Oct-4 expression by RT-PCR. Oct-4 was expressed only in undifferentiated Miz-hES1 cells (not in differentiated Miz-hES1 cells). Lane 1: 100-bp DNA ladder; lane 2: negative control; lane 3: primary mouse embryonic fibroblasts; lane 4: undifferentiated Miz-hES1 cells; lane 5: differentiated Miz-hES1 cells (day 30). The sizes of PCR product of the human ?-actin, mouse b-actin, and human Oct-4 primers are 838, 540, and 445 bp, respectively. (C): Karyotype analysis of hESC lines using G-band method. The karyotype of Miz-hES1 was examined at passage 30 after thawing. The karyotype was found to be normal (46 XY). Abbreviations: hESC, human embryonic stem cell; RT-PCR, reverse transcription–polymerase chain reaction.
Differentiation of Frozen and Thawed hESCs In Vivo
The developmental potential of the addition of type IV collagen or laminin on frozen-thawed hESCs was examined in vivo using a teratoma model. The three hESC lines (Miz-hES1, Miz-hES2, and Miz-hES3 of 44, 51, and 36 passages after thawing, respectively) used in this research formed teratomas after injection into SCID mice. Each teratoma contained representative tissues of the three embryonic germ layers, including endoderm (glandular-like tissue), mesoderm (osteoid-like tissue, cartilage, and adipocyte), and ectoderm (neural tube components). Representative tissues of three germ layers from frozen and thawed hESCs are demonstrated (Fig. 5).
Figure 5. In vivo differentiation of type IV collagen–treated or laminin-treated frozen-thawed human embryonic stem cells. (A): Choroids plexus-like tissue from frozen-thawed Miz-hES1 cells. (B): Cartilage-like tissue from frozen-thawed Miz-hES1 cells. (C): Osteoid- and marrow-like tissue from frozen-thawed Miz-hES2 cells. (D): Neural tube-like tissue from frozen-thawed Miz-hES3 cells. (E):Adipocyte-like cells from frozen-thawed Miz-hES2 cells. (F): Glandular-like tissue from frozen-thawed Miz-hES3 cells. Magnification, x100. Scale bars indicate 100 μm.
DISCUSSION
We thank Dr. Douglas N. Foster at the University of Minnesota and Dr. Seungkwon You at Korea University for proofreading the manuscript. This research was supported by grants M102KL010001-02K1201-00310 and M102KL010001-03K1201-00610 from the Stem Cell Research Center of the 21st Century Frontier Research Program, funded by the Ministry of Science and Technology, Republic of Korea.
REFERENCES
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.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;8:399–404.
Gearheart J. New Potential for human embryonic stem cells. Science 1998;282:1061–1062.
Bongso A, Fong CY, Ng SC et al. Isolation and culture of inner cell mass cells from human blastocysts. Hum Reprod 1994;9:2110–2117.
Freshney RI. Culture of Animal Cells: A Manual of Basic Technique. New York: Wiley-Liss Inc., 1994:255–265.
Reubinoff BE, Pera MF,Vajta G et al. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001;16:2187–2194.
Hawkins AE, Zuckerman MA, Briggs M et al. Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank. J Virol Methods 1996;60:81–88.
Tedder RS, Zuckerman MA, Goldstone AH et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995;46:137–140.
Frederickx V, Michiels A, Goossens E et al. Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells. Hum Reprod 2004;19:948–953.
Woods EJ, Liu J, Pollok K et al. A theoretically optimized method for cord blood stem cell cryopreservation. J Hematother Stem Cell Res 2003;12:341–50.
Xu C, Inokuma MS, Denham J et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotech 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. Nature Biotech 2002; 20:933–936.
Li X, Chen Y, Scheele S et al. Fibroblast growth factor signaling and basement membrane assembly are connected during epithelial morphogenesis of the embryoid body. J Cell Biol 2001;153:811–822.
Kleinman HK, McGarvey ML, Liotta LA et al. Isolation and characterization of Type IV procollagen, laminin, and heparan sulfate proteglycan from the EHS sarcoma. Biochemistry 1982;21:6188–6193.
Ekblom P,Vestweber D, Kemler R. Cell-matrix interactions and cell adhesion during development. Annu Rev Cell Biol 1986;2:27–47.
Li S, Harrison D, Carbonetto S et al. Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation. J Cell Biol 2002;157:1279–1290.
Jaroslaw C, Anna W. Embryonic stem cell differentiation: The role of extracellular factors. Differentiation 2001;68:167–174.
Cooper S, Pera MF, Bennett W et al. A novel keratan sulfate proteoglycan from a human embryonal carcinoma cell-line. Biochem J 1992;286:959–966.
Carpenter MK, Rosler ES, Fisk GJ et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 2004;229:243–258.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation of self-renewal of ES cells. Nat Genet 2000;24:372–376.
Andrews PW, Oosterhuis JW, Damjanov I. Cell lines from human germ cell lines. In: Robertson EJ, ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford, U.K.: IRL Press, 1987:207–24.
Lim JW, Bodnar A. Proteome analysis of conditioned medium from mouse embryonic feeder layers which support the growth of human embryonic stem cells. Proteomics 2002;2:1187–1203.
Zhu X, Assoian K. Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell 1995;6:273–282.
Srebrow A, Friedmann Y, Ravanpay A et al. Expression of Hoxa-1 and Hoxb-7 is regulated by extracellular matrix-dependent signals in mammary epithelial cells. J Cell Biochem 1997;69:377–391.
Burdon T, Smith A, Savatier P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 2002;12:432–438.
Cooper M, Tamura N, Quaranta V. The major laminin receptor of mouse embryonic stem cells is a novel isoform of the a6b1 integrin. J Cell Biol 1991;115:843–850.
XIaofeng L, Ulrika T, Jan T et al. Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane. Proc Natl Acad Sci U S A 2001;98:14416–14421.
Chen Y, Li X, Eswarakumar P et al. Fibroblast growth factor (FGF) signaling through PI3-kinase and Akt/PKB is required for embryoid body differentiation. Oncogene 2000;19:3750–3756.
Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science 2000;287:1427–1430.
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.(Sun Jong Kima,b, Jong Hyu)