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Keywords:

  • embryonic stem cells;
  • human;
  • feeder-free;
  • undifferentiated;
  • Matrgiel™;
  • characterization

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We describe an improved and more robust protocol for transfer and subsequent propagation of human embryonic stem cells under feeder-free conditions. The results show that mechanical dissociation for transfer of the human embryonic stem cells to Matrigel™ resulted in highest survival rates. For passage of the cultures on the other hand, enzymatic dissociation was found to be most efficient. In addition, this method reduces the time, work, and skills needed for propagation of the human embryonic stem cells. With the present protocol, the human embryonic stem cells have been cultured under feeder-free conditions for up to 35 passages while maintaining a normal karyotype, stable proliferation rate, and high telomerase activity. Furthermore, the feeder-free human embryonic stem cell cultures express the transcription factor Oct-4, alkaline phosphatase, and cell surface markers SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and formed teratomas in severe combined immunodeficient mice. This method provides distinct advantages compared with previous protocols and make propagation of human embryonic stem cells less laborious and more efficient. Developmental Dynamics 233:1304–1314, 2005. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Human embryonic stem (hES) cell lines are derived from the inner cell mass of the developing blastocyst and maintained undifferentiated for an extended period of passages while retaining stable karyotype and phenotype (Thomson et al., 1998). The hES cells have the capacity to differentiate into cells and tissues of all three germ layers, both in vivo and in vitro, and are said, thus, to be pluripotent (Thomson et al., 1998; Xu et al., 2001). The unique properties of hES cells suggest that they may supply an almost unlimited source of cells for future replacement therapies, functional genomics, and proteomics as well as for drug screening. Future replacement therapies involving hES-derived cells or tissues will require that the cells and tissues are produced without contact with any animal sources. Furthermore, the use of hES cells also relies on the availability of routine large-scale culturing protocols for undifferentiated hES cells.

Mouse ES cells can be cultured without feeder cells if the medium is supplemented with leukemia inhibitory factor (LIF; Smith et al., 1988; Williams et al., 1988). However, in cultures of hES cells, LIF does not have this effect (Thomson et al., 1998; Reubinoff et al., 2000). Today, the derivation of hES cell lines requires either human or mouse embryonic fibroblast feeders for coculturing (Thomson et al., 1998; Reubinoff et al., 2000; Richards et al., 2002, 2003; Amit et al., 2003; Hovatta et al., 2003). Protocols for the transfer and propagation of hES cultures from feeder to feeder-free conditions have been described previously (Xu et al., 2001; Amit et al., 2004). These feeder-free culture protocols had limitations concerning scale-up properties, low success rate in the initial transfer of the hES cells from feeder to feeder-free conditions (Xu et al., 2001), as well as generating a mixed population of undifferentiated and differentiated hES cells in the cultures (Xu et al., 2001; Amit et al., 2004). A recent study showing contamination of hES cells grown on animal feeders further emphasizes the need for feeder-free protocols (Martin et al., 2005).

In the present study, different techniques for the transfer of hES cells to a feeder-free environment were evaluated, concerning cell adhesion, survival rate, and propagation efficiency. This was done either by mechanical or enzymatic dissociation (Collagenase IV). Furthermore, this culture method was developed to facilitate long-term propagation and large-scale production of homogenous populations of undifferentiated hES cells. The use of conventional cryopreservation techniques for freezing/thawing of the feeder-free hES cultures was also examined.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Four different hES cell lines SA 002, AS 038, SA 121 (Heins et al., 2004), and SA 167 were used in all experiments. The cell lines were propagated on Matrigel™ for up to 35 passages (Table 1), and the morphological appearance and other hES characteristics remained unaltered even after a cycle of freeze/thawing. All cultures consisted of well-defined colonies of hES cells without morphological signs of differentiation (Figs. 1, 2).

Table 1. Summary of the Total Number of Passages for Each of the Four hES Cell Lines (SA 002, AS 038, SA 121, and SA 167) Initially Cultured on mEF Layer Before Transfer to Feeder-Free Propagation on Matrigel
hES cell lines:Number of passages
Cultured on mEFCultured on Matrigel before freezingCultured on Matrigel after thawingTotal
  1. hES, human embryonic stem cells; mEF mouse embryonic feeder cells.

SA 0027161087
AS 0382325654
SA 121246535
SA 167756 + 2442
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Figure 1. a: Nile red–stained clusters of human embryonic stem (hES) cells after mechanical dissociation. Each cluster contains an average of 400–600 cells. b–h: Examples of undifferentiated colony growth for cell line SA 167 cultured on Matrigel for 10 hr (b), 1 day (c), 2 days (d), 3 days (e), 4 days (f), 5 days (g), and 6 days (h) after seeding.

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Figure 2. Colony morphology of undifferentiated colonies of all four cell lines (SA 002, AS 038, SA 121, SA 167) cultured on Matrigel on day 4 after seeding.

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Conditioned Medium

In previous experiments, we have seen that the use of mouse embryonic feeder (mEF) cells up to day 3 (passage two) provides the best conditions for maintenance and proliferation of hES cells in coculture with mEF (data not shown). These observations led to the decision to only use the mEF cells (in passage 2) for up to 3 days, when conditioning the VitroHES medium.

Transfer of hES Cells to Matrigel

To optimize the transfer of the hES cultures from feeder to feeder-free conditions, two different techniques were evaluated; one with mechanical dissociation and one with enzymatic dissociation (Collagenase IV). Mechanical dissociation resulted in a more efficient attachment of cells to the Matrigel and, thus, a more efficient propagation of undifferentiated colonies compared with the enzyme-treated cultures. A significantly larger area (P < 0.001) of surviving colonies was observed 2 days after plating, when mechanical dissociation was compared with enzymatic dissociation (Fig. 3). Furthermore, 6 days after plating, the total colony area in the mechanically dissociated cultures were significantly increased compared with the enzymatically dissociated cultures (P = 0.036; Fig. 3).

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Figure 3. A comparison chart of the two different techniques used for human embryonic stem (hES) cell dissociation (Collagenase and Mechanical dissociation), when transferring the cell lines onto Matrigel. The relative colony area (mm2) was compared between the two different dissociation techniques, on day 2 and day 6 after transfer of the hES cells from mouse embryonic feeder (mEF) cultures to Matrigel.

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Viability Study on hES Cells Dissociated Mechanically Vs. Enzymatically

The cell viability before transfer of the cells to Matrigel was compared between the two different dissociation techniques (n = 12). Enzymatic treatment (Collagenase IV) for 30 min resulted in an average viability of 87.7% (standard deviation, 8.2%) and the mechanical dissociation gave an average viability of 79.5% (standard deviation, 9.6%). The results indicate that cell viability does not influence the propagation efficiency when comparing the two transfer procedures.

Cluster Sizes After Dissociation

The mechanical dissociation protocol resulted in fewer and larger clusters, whereas the enzymatic dissociation produced a cell suspension with a large amount of single cells and many smaller clusters (n = 5). The enzymatic treatment produced an average of 1,850 counts with a size median of 34 μm2 (ranging from 8 to 535 μm2), whereas the mechanical treatment resulted in an average of 160 counts with a size median of 243 μm2 (ranging from 10 to 11,780 μm2).

Passage

Observations were made that, during passage of the hES cells cultured on Matrigel, enzyme treatment with Collagenase IV was needed to detach the colonies from the surface. Mechanical detachment from Matrigel required the use of considerable mechanical force, resulting in individual floating cells, differentiated embryoid body (EB)-like structures, and no analyzable undifferentiated colonies.

Mitotic Index

The mitotic index was calculated to compare the growth rate between feeder-free and mEF cultured hES cells by quantifying the number of cells in mitosis as defined by immunoreactivity for phosphorylated Histone H3. The mitotic index was similar in cultures grown under feeder-free (Matrigel) compared with mEF conditions (Table 2). The doubling time for the feeder-free cultures was roughly the same (approximately 35 hr) as previously reported for feeder-free (Xu et al., 2001; Rosler et al., 2004) and mEF (Amit et al., 2000) propagated hES cells.

Table 2. Mitotic Index for the hES Cell Line SA 121 (Percentage Of Cells In Mitosis)
Culture method:MeanSD
  1. hES, human embryonic stem cells. mEF, mouse embryonic feeder layer.

Feeder (mEF)4.190.939
Feeder-free (Matrigel)3.500.655

Characterization

Pluripotency and maintenance of the four different hES cell lines in feeder-free conditions was demonstrated and compared with previous results for mEF cultures of the respective cell lines (Heins et al., 2004) as well as for SA 167 on mEF cultures (Table 3). These characterizations were performed by examining the morphology, expression of undifferentiated markers, telomerase activity, karyotype, and differentiation in vivo. An overview of the characterization experiments performed on all four hES cell lines after transfer and prolonged propagation on Matrigel is shown in Table 3.

Table 3. Overview of Characterization for All Four hES Cell Lines (SA 002, AS 038, SA 121, and SA 167) After Transfer to and Propagation on Matrigel™
 SA 002AS 038SA 121SA 167
MatrigelaMatrigelaMatrigelamEFMatrigela
  • a

    Characterization of Matrigel cultured cells were performed after a cycle of freeze/thawing.

  • hES, human embryonic stem cells; mEF, mouse embryonic feeder cells; ND, not determined.

SSEA-1
SSEA-3+++++
SSEA-4+++++
TRA 1–60+++++
TRA 1–81+++++
ALP+++++
Karyotyping47 XX46 XY46 XY46 XX46 XX
Fish 13, 18, 21, X, Y+13 XX2n XY2n XY2n XX2n XX
Fish 12, 17q2n XX2n XY2n XYND2n XX
Telomerase activityNDHighHighHighHigh
Oct-4+++++
Teratomas+NDND++
Freeze/Thaw+++++
Immunocytochemistry and RT-PCR.

SSEA-1 expression was negative in all feeder-free cultured hES cell lines as opposed to staining with antibodies against SSEA-3, SSEA-4, TRA-1-60, and TRA 1-80, which show a clear positive immunoreaction (Fig. 4c–f) as expected for pluripotent hES cells. Furthermore, the cells displayed high levels of alkaline phosphatase (AP) reactivity (Fig. 4a), and Oct-4 expression (Fig. 5) was detected by RT-PCR in all four Matrigel propagated cell lines.

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Figure 4. a–f: Examples of staining for alkaline phosphatase (AP) activity and fluorescent immunostaining performed on the undifferentiated cell line SA167 cultured on Matrigel and after a cycle of freeze/thaw: (a) AP, (b) SSEA-1, (c) SSEA-3, (d) SSEA-4, (e) Tra-1-60, (f) Tra-1-81 staining.

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Figure 5. Reverse transcriptase-polymerase chain reaction analysis for Oct-4 expression performed for all four cell lines (SA 002, AS 038, SA 121, SA 167) after transfer to Matrigel and after a cycle of freeze/thaw. The gel is 1.5% agarose, stained with ethidium bromide. 1, 100-bp DNA ladder; 2, cell line SA 002; 3, cell line SA 121; 4, cell line SA 167; 5, cell line AS 038; and 6, negative control (water). Oct-4 polymerase chain reaction product is 247 bp.

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Teratoma formation.

Teratoma formation was performed for two Matrigel cultured hES cell lines, SA 167 and SA 002, and for mEF cultured cell line SA 167 (Table 3). The results showed that teratomas formed consisting of differentiated cells and tissue representative from all three germ layers (endoderm, mesoderm, and ectoderm; Fig. 6), providing evidence that the Matrigel propagated hES cultures have retained their pluripotency. Teratomas were performed previously on the mEF cultured cell lines SA 002, AS 038, and SA 121 (Heins et al., 2004).

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Figure 6. a–d: Teratoma generated from Matrigel cultured cell line SA 002 after injection under the renal capsule in immunodeficient SCID mice showing: teratoma overview (a), ectodermal differentiation, neuroectoderm (b), mesodermal differentiation, cartilage (c), and endodermal differentiation, columnar epithelium with numerous goblet cells (d).

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Karyotyping and fluorescence in situ hybridization analysis.

Karyotype analysis was preformed on mEF and Matrigel cultured cell lines. Three of the cell lines have been characterized previously on mEF cultures (Heins et al., 2004). Furthermore, additional genetic analysis on the cell lines after Matrigel propagation was performed. A total of four karyotypes from SA 002, three karyotypes from AS 038, 10 karyotypes from SA 121, and 17 karyotypes from SA167 was analyzed after prolonged propagation on Matrigel (Fig. 7; Table 3). The results suggested that the hES cell karyotype remains comparable to mEF cultured cells during feeder-free conditions.

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Figure 7. Example of karyotypic analysis performed for cell line SA 121, cultured on Matrigel and after a cycle of freeze/thaw.

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Fluorescence in situ hybridization (FISH) analysis (chromosomes X, Y, 12, 13, 17q, 18, 21) was performed on all of the Matrigel propagated cell lines (Table 3). Additionally, FISH analysis (chromosomes X, Y, 13, 18, 21) was performed on mEF cultured SA 167 cell line. The results show normal presence of the analyzed chromosomes, except for cell line SA 002, which is trisomic for chromosome 13.

Telomerase activity.

Analysis was preformed on three of the Matrigel cultured hES cell lines (AS 038, SA 121, and SA 167). All analyzed hES cells cultured on Matrigel (Fig. 8) were found to have high levels of telomerase activity (Table 3). These results were comparable to the telomerase activity previously published by Heins et al. (2004).

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Figure 8. The relative telomerase activity (RTA), shown for Matrigel cultures of cell line SA 121, AS 038, SA 167, and the negative control, in percentage of the positive control.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Human and mouse ES cells are traditionally cultured on mEF layers (Thomson et al., 1998) but can be propagated in feeder-free environments (Xu et al., 2001; Amit et al., 2004). In this study, we have developed an efficient technique for the transfer of hES cells from feeder to feeder-free culture based on mechanical dissociation. On the other hand, enzymatic dissociation was used for passage of the cultures. The current method leads to higher expansion efficiency combined with markedly improved purity of undifferentiated cells by virtually eliminating the presence of differentiated hES cells from the cultures compared with previously published methods (Xu et al., 2001; Amit et al., 2004). According to the method by Xu et al. (2001), the cultures contain a mixed population of cells where the undifferentiated colonies are surrounded by a layer of differentiated hES cells from the same origin. The pure undifferentiated cultures generated using the present method are more useful for further applications such as genetic analysis (DNA array), differentiation experiments (where uncontrolled spontaneous differentiation could interfere). Further applications are monitoring that cells are maintained in an undifferentiated state using PCR, Western analysis, as well as for creating controlled culture conditions (where differentiated cells may secrete unknown bioactive factors to the medium). The transfer of cells using the mechanical dissociation technique is more efficient in terms of generating larger amounts of cells. The present study, thus, describes a more-efficient method compared with previous protocols (Xu et al., 2001; Amit et al., 2004) and extends the characterization of the feeder-free culture system.

According to the current protocol, the hES cells can also be successfully frozen and thawed using conventional cryopreservation techniques. Furthermore, this method provides a culturing system that more closely resembles conditions used for routine propagation of various cell lines.

Enzyme treatment in the transfer step may negatively affect the cell survival, propagation, and/or differentiation. Therefore, we compared enzymatic treatment with mechanical dissociation when transferring the hES cultures to Matrigel. In the present study, mechanical dissociation was found to be superior to the enzyme treatment regarding success rate, initial adhesion, colony size, and propagation of undifferentiated hES cell cultures. The colony sizes, after using the mechanical dissociation for transfer, was significantly larger on day 2 and 6 after plating, compared with when using the enzymatic dissociation protocol. The colonies were dense with sharp edges and a homogenous, compact morphological appearance, characteristic for undifferentiated hES cell. These features were maintained throughout the entire propagation period on Matrigel.

When transferring the hES cells to Matrigel, the colonies were mechanically cut from the feeder, using only the center part of each colony, whereas in previous work by Xu et al. (2001), entire colonies were detached by enzymatic treatment with the risk of contaminating the cultures with feeder cells. Furthermore, the use of enzymes, at the very delicate step of transferring the feeder cultured hES cells to a feeder-free surface, may cause inactivation of important surface molecules involved in cell adhesion and growth. The major components in Matrigel are extracellular matrix proteins, such as collagen type IV and laminin. Activation of the cell surface integrins upon binding to extracellular matrix proteins is believed to be a crucial step for the regulation of cell adhesion, survival and proliferation. For example, integrin alpha 1 has a unique role among the collagen receptors in regulating both in vivo and in vitro cell proliferation in collagenous matrices (Pozzi et al., 1998). Laminin-specific receptors, possibly formed by integrin α6 and β1, which are highly expressed by hES cells (Xu et al., 2001), may also play a major role in the adhesion of hES cell to the matrix surface. Thus, one possibility is that some of the important surface receptors for attachment or survival might be negatively affected by the rough initial Collagenase IV treatment before the cells have adapted to the new surface.

In the present study, the cell cluster size also proved to be of importance for adhesion, survival, propagation, and maintenance of undifferentiated hES cultures on Matrigel. Large aggregates in the cell suspension tended to form EB-like structures containing differentiated cells, and too small aggregates or single cell suspensions did not support adhesion and survival of the cells. For serial passage on Matrigel, the cluster size was also important but not as crucial as for the transfer step. The average cluster size after enzymatic treatment (used for passage) was generally smaller than after mechanical dissociation, and more single cells were found in the enzyme-treated cell suspensions. The larger cluster size generated by mechanical treatment proved to be favorable at the delicate step of transferring the hES cells to the feeder-free conditions. However, for passage on Matrigel, the enzyme dissociation technique was superior. All our attempts to use mechanical dissociation for passage have failed. Mechanical passaging from Matrigel required the use of considerable mechanical force and resulted in individual floating cells and differentiated EB-like structures but yielded no analyzable undifferentiated colonies. Our conclusion, therefore, is that mechanical dissociation for passaging is inapplicable. The survival of the culture was dependent on the total number of cells seeded, and there appears to be a threshold cell plating density under which cell survival is dramatically impaired.

The quality of the conditioned medium was an important factor for hES cell maintenance on Matrigel. In earlier, nonpublished data, it was noted that mEF cells in passage 2 and no older than 3 days gave the optimal conditions for coculturing with hES cells. An explanation for this finding may be that, after day 3, the ability of the feeder cells to produce or release the undefined factors needed for hES cell survival, proliferation, and maintenance gradually declined. Based on these observations, it was decided to only use the mEF cells in passage 2 for a maximum of 3 days, for conditioning of the VitroHES™ medium.

Studies by Richards et al. (2002) showed that the hES cell lines could not be propagated in an undifferentiated state for more than six passages on cell-free matrixes, including Matrigel. In the present study, however, the hES cells were found to be stable for up to 35 passages on Matrigel, still expressing the markers for undifferentiated ES cells, even after a cycle of freeze/thawing with growth rates remaining roughly comparable. These contradicting results could be due to the fact that all cell lines are unique and behave in different ways. One alternative explanation could also be that the transfer to feeder-free cultures may be more critical than previously thought.

Recently another feeder-free method has been reported (Amit et al., 2004) based on fibronectin matrix and growth factors. The use of human fibronectin matrix instead of Matrigel is advantageous with regard to the source of material, further reducing the risk of contamination with animal pathogens. Although, in the feeder-free work by Amit at al. (2004), there are disadvantages compared with the method described here, namely the generation of a mixed population of differentiated and undifferentiated hES cells in the cultures with a statistically higher differentiation rate and lowered growth rate compared with the feeder cultures. The present method, on the other hand, generates homogenous populations of undifferentiated hES colonies virtually free from contaminating differentiated hES cells, as well as maintaining a growth rate similar to that of feeder cultures.

Slow-rate freezing and rapid thawing methods are most commonly used for cryopreservation of cell lines (Freshney, 1994) and have been shown to be effective for freezing mouse ES cells (Robertson, 1987). This method has not been very successful for freezing hES cell lines, although similar methods have been used (Amit et al., 2003). The more complicated vitrification method has been applied more successfully for freezing of hES cells (Reubinoff et al., 2000, 2001; Richards et al., 2002; Hovatta et al., 2003). However, in the present study, the standardized cryopreservation technique was used and proved to be efficient. The survival rate was high, and no morphological or marker differences could be seen after freezing and thawing. This technique has the advantage to vitrification methods in that the risk of contamination is lowered, as well as being less laborious. In vitrification techniques, colonies are cut and frozen in large pieces compared with the present technique in which a mixture of single cells and small aggregates were frozen. When freezing large aggregates using the present technique, the cells did not survive after thawing (unpublished data). These results suggested that the size of the aggregates were very important for survival after thawing, depending on the freezing technique used.

All the cell lines used in the present study (SA 002, AS 38, SA 121, and SA 167), which were cultured under feeder-free conditions, expressed the POU transcription factor 4 (Oct-4), Alkaline Phosphatase (AP), and surface markers SSEA-3, SSEA-4, Tra 1-60, Tra 1-81. The cells had a doubling time similar to that reported in previous studies for feeder-free culturing of hES cell (Xu et al., 2001). Teratoma formation proved the capacity of the feeder-free propagated hES cells to differentiate to all three embryonic germ layers. The four cell lines used showed similar results in characterization before (Heins et al., 2004) and after the transfer to feeder-free cultures. Thus, proving the maintenance of pluripotency and other hES features when propagated on Matrigel. Recently, Draper et al. (2004) presented data on the gain of chromosomes 12 and 17q in three independent hES cell lines after propagation on feeder and in feeder-free conditions. They suggest that this chromosomal gain may provide a selective advantage for the propagation of undifferentiated hES cells. We, therefore, performed FISH analyses on all our cell lines cultured on Matrigel for chromosomes 12 and 17q without detecting any abnormalities.

In conclusion, the present study describes a robust and versatile method for the transfer of hES cells to feeder-free cultures on Matrigel, where propagation of the hES cells in an undifferentiated state can be carried out without laborious manual cutting and transfer of colonies. This method is fully comparable to feeder culturing with regard to differentiation and growth rates as well as for maintaining all the normal hES cell features. The current protocol facilitates large-scale production of hES cells and will hopefully make hES culturing less dependant of extensive prior experiences. This culturing system can be effectively used for optimization experiments of feeder-free hES cell cultures in the future, regarding for example, medium development, comparative studies of the effect of different substrates, and also facilitate a standardized production of hES cells for various experiments such as animal studies, where large amounts of cells are required.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Human ES Cell Lines

Initially, the hES cell lines used in this study were maintained on a monolayer of Mitomycin C (Sigma Aldrich, Sweden) treated mouse embryonic feeder (mEF) cells (Thomson et al., 1998) for 7 to 71 passages (Table 1), cultured in human embryonic stem cell medium composed as previously described (Xu et al., 2001; Amit et al., 2000), and currently manufactured as VitroHES medium by Vitrolife AB, Kungsbacka, Sweden. Three of the cell lines (SA 002, AS 038, and SA 121) were established, cultured, and characterized by Heins et al. (2004). Briefly, the cell lines used in the present study were established using different techniques and derived from different origins. Cell line SA 002 was established from a blastocyst collected from Sahlgrenska University hospital, Gothenburg, after spontaneous hatching. Cell line AS 38 was established from a blastocyst collected from the Academic hospital in Uppsala, after pronase treatment. Cell line SA 121 was established from a blastocyst collected from Sahlgrenska University hospital, Gothenburg, after pronase treatment followed by immunosurgery. The fourth hES cell line SA 167 has not been published previously. It was established from a blastocyst collected from Sahlgrenska University Hospital, Gothenburg, using enzymatic treatment of the zona pellucida using pronase treatment (Solter and Knowles, 1975) and characterized according to the same criteria as for the other three cell lines (Heins et al., 2004).

Preparation of Conditioned VitroHES-Medium

To prepare mEF cells for conditioning of VitroHES-medium, a confluent monolayer of mEF cells (passage 2) was Mitomycin C treated and seeded at a density of 59,000 cells/cm2 in a gelatin (0.1%; Sigma) -coated culture flask in Dulbecco's modified Eagle medium (D-MEM) supplemented with 1% penicillin/streptomycin (PEST; 10,000 U/ml), 10% fetal bovine serum, and 2 mM GLUTAMAX-I Supplement (200 mM); all from GibcoBRL/Invitrogen (Carlsbad, CA). After a 24-hr incubation period and one wash with phosphate buffered saline (PBS; GibcoBRL/Invitrogen), the medium was discarded and replaced with VitroHES-medium (0.28 ml/cm2) for a 24-hr conditioning period. The conditioned VitroHES-medium (k-VitroHES-medium) was collected every day up to three times from the same mEF culture and sterile filtered by using a 0.2-μm low protein binding filter (Sarstedt, Landskrona, Sweden). The k-VitroHES-medium was used either fresh or after freezing at −20°C and supplemented with 4 ng/ml of basic fibroblast growth factor (bFGF; GibcoRL/Invitrogen) before use. The k-VitroHES-medium may be used for up to 1 week if stored at +4°C. When stored at −20°C for up to 2 months, no sign of reduced bioreactivity could be detected upon usage.

Transfer of hES Cells to Matrigel

BD BioCoat Matrigel Cellware (thin layer, 100 μg/cm2, Becton Dickinson [BD], Bedford, MA) was rehydrated by adding 1 ml of k-VitroHES-medium to each well and incubated for 30 min in 37°C.

Two different techniques were evaluated for the transfer of the hES cells onto Matrigel; one with mechanical dissociation and one with enzymatic treatment. The hES colonies were mechanically cut into small square pieces, representing the center of each colony, by using Stem Cell Tools (Swemed Lab AB, Billdal, Sweden), carefully detached and transferred to Hanks' balanced salt solution (HBSS; Gibco/Invitrogen).

Collagenase IV treatment.

After washing in HBSS, the cell clusters were transferred to a Collagenase IV solution (200 U/ml; Sigma) for enzymatic dissociation. The cells were incubated for 30 min at 37°C and 5% CO2. During the incubation period, repeated mechanical dissociations with a pipette were performed and the dissociation process monitored in an inverted microscope. After the incubation period, the cell suspension was pelleted (400 G for 5 min) and washed once in KnockOut D-MEM (GibcoBRL/Invitrogen) before being resuspended in k-VitroHES medium.

Mechanical dissociation.

After washing in HBSS, the cell clusters were carefully dissociated mechanically by using a 1-ml automatic pipette. The dissociation process was completed when the clusters contained approximately 400–600 cells (Fig. 1a).

After washing and resuspension in k-VitroHES-medium, the cells were transferred to rehydrated Matrigel (six-well thin layer plates; BD) at a cell density of 10 to 15 clusters/well. For the two different transfer techniques, the cells were seeded into four wells each and incubated at 37°C in 5% CO2. Each experiment was repeated four times, with the same amount of cells seeded each time. The number of cells initially used for the two different dissociation protocols were identical. After 2 and 6 days, the colony size and number were calculated. The colony area was calculated by measuring the X and Y diameter of all hES colonies with undifferentiated morphology, allowing for an approximation of cell growth. These colonies consist of a monolayer of homogenously sized cells making this approximation possible. Colony area to cell number correlation was calculated and an approximately linear correlation between colony size and cell number was found in undifferentiated Matrigel cultures. The area/cell relationship averaged at 82 μm2 with individual cell areas ranging from 30 to 134 μm2.

Viability Study on hES Cells Dissociated Mechanically Vs. Enzymatically (Collagenase IV)

The viability test was performed by using the Calcein/Ethidium Homodimer (Calcein/EthD) kit on mEF cultures of hES cells. A comparison was preformed of the hES cells dissociated mechanically vs. enzymatically (see methods above). The dissociated cells were resuspended in 100 μl of Calcein/EthD solution, respectively, and incubated for 10 min in room temperature. The Calcein/EthD solution consisted of EthD stock solution (0.5%), Calcein stock solution (0.25%), and D-PBS (99.25%). Each cell suspension (10 μl) was placed on a glass slide, covered with a cover glass. The dead and live cells were counted by using a microscope (Nikon Eclipse TE2000-U).

Cluster Sizes After Dissociation

Cell line SA 002 cultured on mEF was used for evaluation of cluster size. Colonies were dissociated mechanically and enzymatically (as described above) from equally sized undifferentiated colonies. Suspensions of cell clusters were then incubated with Nile red staining solution (1 μM in PBS) for 10 min and photodocumented using a fluorescence microscope (Nikon Eclipse TE2000-U; Fig. 1a). All clusters were counted and measured using ImageJ image analysis software (ImageJ 1.32, NIH, Bethesda, MD).

Passage of Matrigel Propagated hES Cells

The cell cultures were observed visually by using an inverted microscope (Nikon Eclipse TE2000-U). When ready for passage, the medium was aspirated and 1 ml of Collagenase IV (200 U/ml) solution was added to each well and incubated for 15 to 20 min. To facilitate cell detachment from the surface, careful mechanical dissociation was performed followed by another 15 min of incubation. The cells were then washed, resuspended in k-VitroHES-medium, and seeded at a split ratio of 1:3 to 1:6 onto Matrigel. The hES cultures were passaged every 5 to 6 days, and the medium was changed every second to third day.

Cryopreservation of Matrigel Cultured hES Cells

All cell lines were dissociated as previously described before freezing. After washing and centrifugation, the cells were transferred to a freezing medium, consisting of k-VitroHES medium supplemented with 10% dimethylsulfoxide, 4 ng/ml bFGF, and Serum Replacement (SR) to a final concentration of 30% (all from GibcoBRL/Invitrogen). The cell density was approximately 1 million cells per ml freeze medium. The final cell suspension was a mixture of both single cells and small cell clusters. Nunc CryoTubes (Nalge Nunc International, Rochester, NY) containing 0.5 to 1.0 ml of cell suspension, were rapidly transferred to a Nalgene freezing container (Nalge Nunc International) for storages in −80°C overnight or at least for 4 hr before moved to long-term storage in Liquid Nitrogen.

To thaw the cells, k-VitroHES-medium was prepared and preheated to 37°C before thawing the cells by rapidly transferring the cryotubes to a 37°C water bath until all of the cell suspension was thawed. The cell suspension was then transferred to the preheated k-VitroHES medium, for 5 min equilibration, before centrifugation (400 × g in 5 min). The cell pellet was washed once, resuspended in k-VitroHES-medium and seeded into either 24- or 6-well Matrigel plates at the same cell density as before freezing.

Characterization

Previously defined criteria (Thomson et al., 1998; Heins et al., 2004; Carpenter et al., 2004; Rosler et al., 2004) for in vitro and in vivo characterization were used to demonstrate pluripotency and immortality of the four different hES cell lines used in this study. All characterization experiments on Matrigel cultured cells were performed after a cycle of freeze/thaw. Three of the cell lines used here were characterized previously on mEF cultures and the results published in Heins et al. (2004). The basic characterization of cell line SA 167 has not been published previously, and SA 167, thus, represents a newly established hES line.

Immunocytochemistry.

The feeder-free hES cultures were passaged as described above seeded onto 6- or 24-well Matrigel plates and cultured for 6 days before performing the immunostaining. The cultures were washed in PBS, fixed with 4% formaldehyde (HistoLab, Gothenburg, Sweden) for 15 min at room temperature, and then washed again three times in PBS. The monoclonal primary antibodies used were directed against SSEA-1, -3, and -4 (1:200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA); Tra-1-60, Tra-1-81 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA); and polyclonal rabbit anti-phospho-Histone H3 (1:150; KeLab, Upstate). The primary antibodies were incubated overnight at 4°C before being visualized using appropriate Cy3- or fluorescein isothiocyanate–conjugated secondary antibodies (1:300; Jackson ImmunoResearch Laboratories, West Grove, PA). Cultures were also incubated with 4′-6′diamidino-2-phenylindole (Sigma-Aldrich Sweden AB, Stockholm, Sweden), at a final concentration of 0.5 μg/ml for 5 min at room temperature, to visualize all the cell nuclei. The stained cultures were rinsed and mounted using DAKO fluorescent mounting medium (Dakopatts AB, Älvsjö, Sweden) and visualized in an inverted fluorescent microscope (Nikon Eclipse TE2000-U). AP staining of the Matrigel cultured hES cells was carried out according to the manufacturer's instructions using a commercially available kit (Sigma-Aldrich).

Telomerase activity.

mEF and Matrigel cultured hES cells were harvested and lysed, and telomerase activity was analyzed by a PCR-based enzyme-linked immunosorbent assay (ELISA; Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions.

Karyotyping and FISH.

The hES cells designated for karyotyping were incubated for 1 to 3 hr in Colcemid (0.1 μg/ml, Invitrogen, Carlsbad, CA), dissociated, fixated, and mounted on glass slides, and the chromosomes visualized by using a modified Wrights staining (#WS-32, Sigma). Preparation of metaphase plates was performed as previously described (Karhu et al., 1997; Henegariu et al., 2001). For the FISH analysis, a commercially available kit (MultiVysion PB Multicolour Probe Panel; Vysis, Inc., Downers Grove, IL) containing probes for chromosome 12, 13, 17q, 18, and 21 and the sex chromosomes (X and Y) was used according to the manufacturer's instructions. Slides were analyzed using an inverted microscope equipped with appropriate filters and software (CytoVision, Applied Imaging, Santa Clara, CA).

Teratomas.

For the teratoma formation experiment, immunodeficient SCID mice (C.B-17/lcrCrl-scidBR, Charles River Laboratories, Germany) were used. Matrigel propagated hES colonies were enzymatically detached from the surface by using Collagenase IV (200 U/ml), mechanically dissociated into small cell aggregates and approximately 50,000 to 100,000 cells/organ were injected under the kidney capsule. Control animals were treated with Cryo-PBS injections or with primary brain cells from a littermate. The animals were killed 8 weeks after injection, and the tumors were immediately fixed in a 4% solution of paraformaldehyde and embedded in paraffin. For histological analysis, the teratoma were sectioned to 8 μm and stained with Alcian blue/Van Giesson's stain.

RT-PCR analysis of Oct-4 expression.

Total RNA was isolated from all four Matrigel cultured hES cell lines by using RNeasy Mini Kit (Qiagen) according the manufacturer's instructions. The cDNA was synthesized from 1 μg of total RNA using AMV First Strand cDNA Synthesis Kit (Roche), and the PCR reaction was preformed by using Platinum Taq DNA Polymerase (Invitrogen). The PCR reaction included four initial step-down cycles, with two repeated cycles for every annealing temperature, with denaturation for 15 sec at 94°C, annealing temperature for 15 sec at 66° to 60°C and extension for 30 sec at 72°C. The following cycles included 35 repeats with annealing temperature at 58°C. The forward and reverse primer sequences for Oct-4 were described previously (Nichols et al., 1998). The β-actin primers were used as internal controls (sense, 5′-TGGCACCACACCTTCTACAATGAGC-3′; antisense, 5′-GCACAGCTTCTCCTTAATGTCACGC-3′; 400-bp product). The PCR products were size fractioned by gel electrophoresis using a 1.5% agarose gel. Human liver was used as a positive control and water as a negative control for the PCR reaction.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors thank Mikael Englund and Cecilia Sjöblom for their hard work and expertise regarding the teratoma experiments, Gunilla Caisander for conducting the karyotype analysis, and Katarina Andersson for performing the PCR reactions.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES