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Keywords:

  • Human embryonic stem cells;
  • Pluripotency;
  • Activin A;
  • Defined medium

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Summary
  7. Acknowledgements
  8. References

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain “stemness” in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self-renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct-4, nanog, and TRA-1-60 and remained pluripotent, as shown by the in vivo formation of teratomas.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Summary
  7. Acknowledgements
  8. References

Pluripotency of human embryonic stem cells (hESCs) is maintained when the cells are grown on mouse embryonic feeder layers (mEFs), [1, 2] on laminin or matrigel supplemented with conditioned medium (CM) from mEFs [3] or on human feeder layers [4]. Additionally, signals received from the feeder layers do not operate through the leukemia inhibitory factor (LIF)/gp130 pathway [5, 6], as is the case with mouse embryonic stem cells (mESCs). Consequently, alternate signaling pathways, activated by the contact of hESCs to feeder layers and/or soluble factor(s) present in the conditioned medium, mediate the maintenance of pluripotency. Previously, we showed that the growth and morphology of the hESC line human skin fibroblast (HSF6) were similar when grown on either mEFs or on laminin supplemented with conditioned media from mEFs [6]. HSF6 cells grown on laminin in the absence of mEF-conditioned medium rapidly lose the pluripotency markers Oct-4, nanog, and TRA-1-60, indicating loss of “stemness.” Thus, the conditioned medium contains soluble factors secreted by the feeder layers that are instrumental in maintaining pluripotency. In the study reported here, we showed that hESCs grown on laminin in the presence of activin A, nicotinamide (NIC), and keratinocyte growth factor (KGF) remain undifferentiated during continuous growth over 20 passages.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Summary
  7. Acknowledgements
  8. References

ESC Culture

hESC line HSF6 was maintained on mitomycin-C–treated CF1 mouse feeder layers (mEFs) at 37°C, 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) serum replacement (DSR), as previously described [6]. For the experiments described here, passage 43 hESCs were cultured on dishes coated with laminin (20 μg/ml; Chemicon International, Temecula, CA, http://www.chemicon.com) in the presence of CM from mEFs supplemented with 10 ng/ml basic fibroblast growth factor (bFGF2; PreproTech Inc., Rocky Hill, NJ, http://www.preprotech.com), or on laminin in DSR medium containing 50 ng/ml human recombinant activin A, 50 ng/ml human recombinant KGF (both from PreproTech), and 10 mM NIC (Sigma Chemical Corp., St. Louis, http://www.sigma-aldrich.com). A dose response with hESCs using activin A at 5, 50, and 100 ng/ml showed 50 ng/ml to be optimal to maintain the cells in an undifferentiated state.

In some experiments, 10 ng/ml human recombinant bone morphogenetic protein 4 (BMP-4; R&D Systems, Minneapolis, http://www.rndsystems.com) was used to replace activin A, and 2 μg/ml recombinant mouse FS-288 follistatin (R&D Systems) was added to ESCs grown on mEFs, sufficient to neutralize 50 ng/ml activin A, according to the manufacturer's directions. The medium was changed every day on cells grown on mEFs or in CM and every other day on cells grown on laminin with the growth factors. Cells were passaged weekly at 1:3 or 1:4 dilution by gentle treatment with 1 mg/ml collagenase IV (Gibco BRL, Carlsbad, CA, http://www.invitrogen.com) for 5 minutes, followed by scraping.

Proliferation Assay to Quantify Rates of Proliferation Under Different Culture Conditions

Cells were cultured in six-well plates on laminin in the presence of either bFGF2-supplemented CM from feeder layers or activin A, KGF, NIC, or a combination of these three factors. Cultures were pulsed with 1 μCi/ml [methyl 3H] thymidine (specific activity 6.7 Ci/mmol; MP Biomedicals, Irvine CA, http://www.mpbio.com) in newly replenished medium. After 16 hours, cells were harvested and thymidine incorporation into cells was quantified, as previously described [7]. Briefly, DNA content was measured fluorometrically, and incorporation of 3H thymidine was determined by liquid scintillation counting of trichloroacetic acid precipitates of the sonicated cells. Statistical significance of observed differences was determined by analysis of variance and Fischer's protected least significance difference test with a 95% level as the limit of significance using Statview IV (Abacus Concepts, Berkeley, CA, http://www.abacus.com).

Immunohistochemistry

hESC cultures were grown on coverslips coated with mEFs or laminin, fixed with 4% paraformaldehyde, and immunostained, as previously described [6]. Protein expression of the stem cell markers TRA-1-60, SSEA-4, and Oct-4 was analyzed using primary mouse anti-TRA-1-60 immunoglobulin-M (IgM; Chemicon) mouse anti-SSEA-4 IgG-3 (DSHB; University of Iowa, Iowa City, IA), and rabbit anti-Oct-4 antiserum (a generous gift from Dr. Hans Scholer, University of Pennsylvania). Control slides were incubated with mouse IgM or IgG and rabbit IgG.

Flow Cytometric Analysis to Quantify Undifferentiated Cells Under Different Culture Conditions

Cells were harvested using a 60-minute collagenase treatment, followed by shearing into single-cell suspensions and filtering using a 70-3 cell filter. Single cells from each condition were labeled with mouse anti-TRA-1-60 or mouse IgM (for controls) and fluorescein isothiocyanate (FITC)–conjugated donkey anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). Cells were analyzed using a Becton, Dickinson FACScan (Franklin Lakes, NJ, http://www.bd.com), and cell-surface antigen expression was quantitated using CellQuest software (Becton, Dickinson).

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RNA was purified using the RNeasy minikit including DNase treatment (Qiagen, Valencia, CA, http://www1.qiagen.com) and reverse transcribed using avian myeloblastosis virus (AMV) with 3.2 μg of random primer (both Roche, Indianapolis, http://www.roche-applied-science.com) and 1 μg of total RNA in a reaction volume of 20 μL. 1 μL of cDNA was used for each PCR reaction in a total volume of 50 μL. See Table 1 for specific primers designed. The PCR products were loaded onto a 1.2% agarose gel (1.6% gel for human telomerase reverse transcriptase [hTERT]) and stained with ethidium bromide.

Table Table 1.. Reverse transcription polymerase chain reaction (RT-PCR) primers
GeneProduct (bp)Primer sequencesAccession no.
Oct-43105′-GAGCAAAACCCGGAGGAGT-3′ forward 5′-TTCTCTTTCGGGCCTGCAC-3′ reverseGI: 22056734
nanog2555′-GCTTGCCTTGCTTTGAAGCA-3′ forward 5′-TTCTTGACTGGGACCTTGTC-3′ reverseGI: 13376297
activin A2625′-CTTGAAGAAGAGACCCGAT-3′ forward 5′-CTTCTGCACGCTCCACTAC-3′ reverseGI: 31566358
neuro-D5505′-GAGACTATCACTGCTCAGGA-3′ forward 5′-GATAAGCCCTTGCAAAGCGT-3′ reverseGI: 4587071
Brachyury T2905′-CAACCACCGCTGGAAGTAC-3′ forward 5′-CCGCTATGAACTGGGTCTC-3′ reverseGI: 2558580
α-fetoprotein [17]6805′-AGAACCTGTCACAAGCTGTG-3′ forward 5′-GACAGCAAGCTGAGGATGTC-3′ reverseGI: J00077
Activin receptors:   
    ALK-4 (type I)3465′-CACGTGTGAGACAGATGGG-3′ forward 5′-GGCGGTTGTGATAGACACG-3′ reverseGI: 402188
    ACVR-2 (type II)7835′-GGGAGCTGCTGCAAAGTTG-3′ forward 5′-CCACATCAACACTGGTGCC-3′ reverseGI: 10862696
    ACVR-2B (type II)6115′-CACCATCGAGCTCGTGAAG-3′ forward 5′-GAGCCCTTGTCATGGAAGG-3′ reverseGI: 10862697
hTERT1145′-CAGCTCCCATTTCATCAGCA forward 5′ CGACATCCCTGCGTTCTTG 3′ reverseGI: 4507438
ß-actin2035′-CGCACCACTGGCATTGTCAT-3′ forward 5′ TTCTCCTTGATGTCACGCAC-3′ reverseGI: 33878222

Two-Dimensional Electrophoresis

Unfractionated conditioned media from mEFs grown alone or as a feeder layer with hESCs was assayed by isoelectric focusing and electrophoresis in the second dimension, as previously described [8]. The sample was transferred to nitrocellulose and blotted with rabbit anti-porcine activin antibody (a generous gift from Dr. Sunichi Shimisaki, University of California, San Diego).

Western Blotting for Phosphorylated Smad2

Western blotting for phosphorylated Smad2 (Homo sapiens MAD, mothers against decapentaplegic homolog 2) was performed on lysates of HSF6 cells, as previously described [6]. Cells cultured in the presence of activin A were lysed in detergent containing buffer supplemented with vanadate (10 μM) and microcystin (1 μM) and first blotted with phospho-Smad2 (ser465/467) antibody (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com). Blots were then stripped and reprobed with Smad2 antibody (Cell Signaling Technology).

Pluripotency

Pluripotency was assessed in vivo by examining teratoma formation 8 weeks after transplanting the hESCs under the renal capsule of nude mice, as previously described for analysis of endocrine pancreatic progenitor cell differentiation [9]. Grafts were removed, fixed, and stained with hematoxylin and eosin. Pluripotency was assessed in vitro by analyzing gene expression in embryoid bodies derived from hESCs and cultured for 17 days.

Karyotype Analysis

Karyotype was analyzed in our laboratory or in the Cytogenetics Laboratory, University of California, San Diego, using standard methods (G-banding). At least 15 cells were examined from each sample.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Summary
  7. Acknowledgements
  8. References

In initial experiments, we sought to develop media to culture hESCs that would direct differentiation into pancreatic endocrine lineage. For cell adhesion we used laminin 1, based on the high levels of α6αß1 expression in hESCs [3]. A cocktail of various growth factors and chemicals previously shown to modulate cellular growth and differentiation in human fetal pancreatic cells was tested. Surprisingly, hESCs cultured for several weeks under these conditions showed no change in cell morphology. Subsequently, each factor was sequentially eliminated and pluripotency was assessed by the expression of known markers for human stem cells: TRA-1-60, nanog, and Oct-4 (data not shown). When the combination of growth factors and chemicals that maintained hESCs replicating in an undifferentiated state was narrowed to activin A (A), NIC (N), and KGF (K), the experiments were repeated with each of these growth factors alone or in various combinations. Staining of the cultures containing all three factors (ANK) was uniform for the stem cell markers TRA-1-60 and Oct-4 (Fig. 1A, panel II), and comparable to the staining for cells on feeder layers (Fig. 1A, panel I) or in CM from mEFs (not shown). Robust gene expression of Oct-4 and nanog was also observed by RT-PCR in the cell monolayers, with levels comparable to those obtained in colonies growing on feeder layers (Fig. 1B). A hallmark of stem cells and “stemness” is clustered growth. Interestingly, hESC appearance gradually changed from the usual tight colony formation to an irregular monolayer of uniformly shaped cells. The cells appeared larger than those observed in the original colonies (Fig. 1A, panel II). With continuous growth, they eventually formed a continuous monolayer and mounded up in the dish. However, these changes were reversible; when cells were placed back on feeder layers, they gradually resumed the colony formation similar to that previously observed on feeder layers (Fig. 1A, panel III).

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Figure Figure 1.. Differentiation of human embryonic stem cells (hESCs) in the absence of activin A. (A): Morphology and differentiation state of human skin fibroblast (HSF6) cells observed by phase contrast microscopy (upper layer) and immunohistochemistry (lower layer). For immunohistochemical analysis, antibodies against the human stem cell markers TRA-1-60 (red, cytoplasmic) and Oct-4 (green, nuclear) were used. Panel I: HSF6 cells cultured on mouse embryonic feeder layers (mEFs) show typical colony formation, with uniform staining for stem cell markers. Panel II: HSF6 cells cultured on laminin in the presence of activin A, nicotinamide (NIC), and keratinocyte growth factor (KGF)—ANK—grow as irregular monolayers, with larger cell size than when grown on mEFs, but show robust staining for TRA-1-60 and Oct-4, proof of their undifferentiated state. Panel III: When put back on mEFs, cells from panel II resume colony morphology after 1 week. Panel IV: Cells from panel II, grown in the absence of activin A (NK) for 1 week, show distinct change in morphology and phenotype, with no staining for TRA-1-60 and very little staining for Oct-4, indicating differentiation. Panel V: Cells from panel II grown in the absence of KGF and NIC for 1 week (A), show no change in phenotype; however, proliferation was reduced and they could not be passaged further. Magnification bar: 100 μM. (B): Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR; 26 cycles) of hESCs for Oct-4 and nanog under a variety of culture conditions on mEFs (lane 1) or on laminin (lanes 2–5). Expression of stem cell markers was lost in cells cultured for 1 week on laminin in the absence of activin (lanes 3, 5), indicating the need for activin to maintain the undifferentiated phenotype. (C): Representative comparison of cell-surface antigen expression using fluorescence-activated cell sorter (FACS) analysis. Single-cell suspensions from different culture conditions were immunostained for TRA-1-60 and analyzed using a Becton, Dickinson FacScan and CellQuest software. Upper panel: Flow cytometric analysis of cells cultured with ANK stained with mouse anti-TRA-1-60 or mouse IgM (control). Lower panel: Comparison of percentage of cells expressing TRA-1-60 under different conditions. Cells were cultured with ANK for 20 passages, with NK for 1 week, and in CM for <1 week (to remove contaminating mEFs). (D): Representative comparison of proliferation of hESCs in the presence of activin A (A), NIC (N), KGF (K), or a combination of all three (ANK), and in basic fibroblast growth factor–supplemented CM. Quadruplicate wells for each condition were pulsed for 16 hours with 3H thymidine. Proliferation rate was significantly lower in activin-treated cells than in all other treatments, and proliferation rate in the presence of KGF was similar to ANK and significantly higher than CM, indicating a role for KGF in replication of hESCs. n = 4, p < .0001 ANK vs. A, K vs. A, CM vs. A; p < .005 ANK vs. N, N vs. A, K vs. N; p < .05 K vs. CM; n.s. ANK vs. K, ANK vs. CM, N vs. CM. Abbreviations: A, activin A alone; ANK, activin A plus NIC plus KGF; CM, conditioned medium; NK, NIC plus KGF; NK + BMP, NK plus bone morphogenetic protein 4; +/−, RT.

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Removal of activin from the growth medium resulted in the rapid change in cell morphology to a differentiated phenotype (Fig. 1A, panel IV); after 1 week without activin A (NK) the cells no longer expressed nanog (Fig. 1B), with concomitant loss of immunoreactive TRA-1-60 (Fig. 1A, panel IV) and reduced levels of Oct-4 protein (Fig. 1A, panel IV) and message (Fig. 1B). The immunohistochemical and RT-PCR data were validated by quantitation of cell-surface antigen expression by flow cytometry. Consistent with a previous report of TRA-1-60 expression in the ESC lines H7 and H14 [10], 60.3% of HSF6 grown on laminin in the presence of mEF conditioned medium expressed TRA-1-60. Of cells grown in the defined medium, 45.96% expressed the antigen at passage 2, 60.46% at passage 10, and 71.9% at passage 20, a similar pattern of expression to the parent cells (Fig. 1C). In contrast, when activin was removed from the culture for 1 week, the level of expression was reduced to 3.9% (Fig. 1C). Cells cultured in the defined growth medium were examined for markers of pluripotency up to and including passage 20 and found to express all markers tested: TRA-1-60, SSEA-4, Oct-4 (immunohistochemical analysis), and Oct-4, nanog, and hTERT (Fig. 2A).

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Figure Figure 2.. Long-term maintenance of pluripotency in human embryonic stem cells (hESCs) cultured with activin A, nicotinamide (NIC), and keratinocyte growth factor (KGF). (A): Analysis of stem cell markers in human skin fibroblast (HSF6) cells cultured in the presence of activin A, NIC, and KGF for 20 passages. Upper panel: Immunohistochemical analysis shows robust staining for TRA-1-60, SSEA-4 (red), and Oct-4 (green). Magnification bar: 200 μM. Lower panel: Reverse transcription polymerase chain reaction (RT-PCR) analysis for Oct-4, nanog (26 cycles), and human telomerase reverse transcriptase (hTERT; 35 cycles, product = 114 bp). For comparison, cells cultured on mouse embryonic feeder layers (mEFs) for a comparable number of passages were analyzed in the same assay, indicating comparable levels of expression of all markers. (B): Teratoma formation in nude mice. Representative histology of HSF6 cells cultured in the presence of activin A, NIC, and KGF transplanted under the renal capsule of nude mice. After 8 weeks, kidneys were removed, and teratomas showing evidence of all three cell layers were observed. Magnification bar: 100 μM. (C): RT-PCR analysis of lineage-specific markers in embryoid bodies derived from hESCs cultured in the presence of activin A, NIC, and KGF shows RNA expression of all cell types. Abbreviations: C, chondrocytes (mesoderm); α-FP, endoderm; neuro-D, ectoderm; PNC, perineural (Schwann) cells (ectoderm); RE, respiratory epithelium (endoderm); T gene, mesoderm [5]; +/−, RT.

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Removal of KGF and NIC from the medium had a different effect: the cells maintained their undifferentiated phenotype (Fig. 1A, panel V, and Fig. 1B). However, there was a significant difference in cell proliferation when cultured with activin, KGF, or NIC alone compared with the combination of the three factors (ANK). Cells cultured with activin alone did not differentiate (A in Fig.1A, panel V, and Fig. 1B); however, their proliferation rate was significantly reduced from those cultured with the combination (ANK) or with KGF or NIC alone (Fig. 1D; n = 4, p < .0001) versus ANK or KGF (p < .005 versus NIC). In contrast, there was no statistical difference in the proliferation rate of cells cultured with KGF, compared with cells cultured with the combination. Proliferation of cells cultured with NIC alone was intermediate; although it was significantly less than that in the KGF- or ANK-treated cells (Fig. 1D; n = 4, p < .005), the rate was significantly higher than in activin-treated cells (Fig. 1D; n = 4, p < .005). From these data we conclude that activin is needed for maintenance of pluripotency and that KGF and, to a lesser extent, NIC help maintain proliferation and continued growth. While cells cultured with activin and KGF (AK) in the absence of NIC remained undifferentiated and grew successfully in the short term, their growth was suboptimal over several passages compared with those cultures that included NIC (data not shown), possibly due to its documented anti-apoptotic effect [11]. Therefore, NIC was included in the growth factor combination used during the 20 passages. Importantly, in addition to maintaining markers of undifferentiated cells for 20 passages, these cells also retained a normal karyotype (data not shown).

We next explored whether another member of the transforming growth factor-ß (TGF-ß) superfamily could maintain pluripotency. Like activin, bone morphogenetic proteins (BMPs), are secreted proteins that regulate numerous cellular responses [12], including differentiation of hESCs into trophectoderm [13]. In addition to its role in differentiation, BMP-4 has also been shown to maintain pluripotency in mESCs [14]. In contrast, when activin A was replaced with BMP-4 in the medium, the hESCs were unable to maintain their undifferentiated phenotype and a complete loss of expression for nanog and Oct-4 occurred after 1 week (Fig. 1B). In mESCs BMP-4 has a paradoxical role in both maintenance of pluripotency and differentiation [14, 15], most likely due to interactions with other growth factors present at particular stages of development and to different concentrations of the peptide to which the cells are exposed [16]. A similar situation may occur with activin A and hESCs, as activin A–induced differentiation of hESCs under certain conditions has already been shown [17]. A recent report from Amit et al. [18] demonstrated successful maintenance of pluripotency in hESCs using TGF-ß1. It is not surprising that activin A and TGF-ß1 have similar effects on hESCs since they act through the same Smad pathway (AR-Smads), while BMPs act through the other major Smad pathway (BR-Smads) [19].

Activin A has been identified in a wide variety of tissues as an autocrine or paracrine regulator of diverse biological functions [20, 21]. Significantly, we found high expression of activin A transcripts in mEFs and secreted activin A precursor protein in the conditioned medium from mEFs (Fig. 3C). In addition, HSF6 cells express high levels of both type I and type II activin receptors and robust Smad2 phosphorylation (Fig. 3D). Moreover, after 2 weeks in the presence of the activin inhibitor follistatin, the HSF6 cells grown on mEFs differentiated, completely losing the ESC markers TRA-1-60, Oct-4, and nanog (Fig. 3A–B), similar to the effect seen with removal of activin A from the defined medium (Fig. 1A–B). FS-288, the isoform of follistatin used in these experiments, has extremely high affinity for activin A, has a lower affinity for members of the BMP family, and does not bind TGF-ß [21, 22]. As we have already shown that BMP-4 is ineffective in maintaining pluripotency in hESCs, it is likely that the differentiation we see results from specific interactions between activin and follistatin.

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Figure Figure 3.. The effect of activin and follistatin (FOL) on mouse embryonic feeder layer (mEF) maintenance of pluripotency in human skin fibroblast (HSF6) cells. (A): HSF6 cells from Figure 1, panel I, cultured on mEFs in the presence of FOL for 1 week (left panels) and 2 weeks (right panels). After 1 week, colonies showed distinct morphologic changes (upper panel) and lost staining for TRA-1-60 (red), with reduced staining for Oct-4 (green) (lower panel). After 2 weeks in the presence of FOL, colonies continued to grow but lost their defined shape and Oct-4 immunoreactivity had completely disappeared, indicating differentiation. Magnification bar: 100 μM. (B): Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR; 26 cycles) of HSF6 cells on mEFs for Oct-4 and nanog in the presence and absence of FOL. Expression of stem cell markers was absent (nanog) or markedly diminished (Oct-4) in cells cultured for 1 week on mEFs in the presence of FOL and completely lost after 2 weeks culture, indicating differentiation. (C): Identification of activin A transcripts in mEFs derived from CF-1 mice and precursor protein in mEF-conditioned medium using RT-PCR and western blots. Left panel: RT-PCR showing activin A expression; PCR product size is 262 bp; +/−: reverse transcriptase. Right panel: Western blot showing activin A precursor protein. Samples were analyzed by two-dimensional electrophoresis, then western blotted using anti-activin antibodies. (D): Identification of activin pathway signaling components in HSF6 cells. Left panel: Type 1 receptor ALK-4 and type II receptors ACVR-2 and ACVR-2B transcripts in HSF6 cells. PCR product sizes are 346 bp, 783 bp, and 611 bp, respectively; +/−: reverse transcriptase. Right panel: Western blot using anti-Smad2 antibodies showing phospho-Smad2 in HSF6 cells grown in the presence of activin A. Smad2 molecular weight, 60 kDa. Cells were lysed in detergent-containing buffer supplemented with vanadate (10 μM) and microcystin (1 μM). Blots were probed with anti-phospho-Smad2 (ser/465/467) (panel I), then stripped and reprobed with anti-Smad2 (panel II).

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Activin A has been implicated in differentiation of mES into mesoderm [15], differentiation of human pancreatic precursor cells into ß cells [23], inhibition of neural differentiation [24, 25], and, more recently, induction of endoderm in hESCs [26]. This, however, is the first documentation of the presence of activin in conditioned medium from mEFs, and its subsequent novel role in the maintenance of stem cells in the undifferentiated state. At this time we do not know the specific target genes for the activin A signaling pathway in hESCs, but it has been shown that there is crosstalk between the TGF-ß/activin and Wnt pathways involving the AR-Smads and LEF1/TCF (lymphoid enhancer binding factor 1/T-cell–specific factor) [27]. We have detected expression of several Wnts in hESCs (data not shown). Therefore, our findings, taken with the recent report of maintenance of hESC pluripotency through activation of Wnt signaling [5], may help elucidate a defined molecular pathway for maintenance of pluripotency in hESCs.

Further proof of the pluripotency of the hESCs maintained in activin A–enriched medium was provided by teratoma formation in vivo. After transplantation of the hESCs under the kidney capsule in nude mice, the grafts showed evidence of ectodermal, endodermal, and mesodermal structures (Fig. 2B). In addition, lineage-specific gene-expression profiles obtained by RT-PCR on 17-day-old embryoid bodies derived also from cells cultured in the presence of activin A showed a similar pattern of expression for all three embryonic cell layers (Fig. 2C). These data show that maintenance of hESCs in medium containing activin A allows the maintenance of pluripotency without the need for coculture with other foreign or human cells.

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Summary
  7. Acknowledgements
  8. References

The identification of activin A as a key factor in mediating these cellular events will help to unravel the biochemical pathways responsible for “stemness.” An increased efficiency in the generation and culture of human stem cells for potential clinical applications is timely, given the recent report of 17 newly derived stem cell lines available for non-federal supported research [28]. The findings reported here may facilitate the derivation of new human embryonic cell lines without the use of animal or human feeder layers.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Summary
  7. Acknowledgements
  8. References

This work was supported by a network grant from the Larry L. Hillblom Foundation. A. Hinton was supported by NIH training grant no. t32 h107276 in molecular medicine and atherosclerosis. We thank Dr. Stephen Baird (University of California, San Diego) for assistance in analysis of teratoma tissue.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Summary
  7. Acknowledgements
  8. References