| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biotechnology, Institut für Tierzucht, Mariensee, D-31535 Neustadt, Germany
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
assisted reproductive technology, early development, embryo, environment, fibroblasts, germ line, Oct4, pluripotency, stem cells
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Fibroblasts are the principal cell type in connective (mesodermal) tissue, where they mediate structural and functional processes, such as formation of a collagen meshwork and wound healing [13–15]. Primary fibroblasts can be obtained from subdermal and other connective tissues [16, 17]. These cells represent a poorly characterized mixture of cell lineages [18], in which the composition varies with the developmental stage of the donor and the tissue of origin [14]. The regenerative ability of connective tissue suggests the presence of tissue-specific progenitor cells.
In the present study, we have tested whether primary fibroblast cultures contain stem cells with the potential for multilineage differentiation. To identify rare stem cells within a large population of primary somatic cells, we used the OG2 transgenic mouse line, which carries the Oct4 (also known as Pou5f1) promoter driving a green fluorescent protein (GFP) marker [19, 20]. Oct4 is a transcription factor of the POU family that is crucial for maintenance of embryonic stem cells in the undifferentiated state. It plays a major role in mouse embryogenesis [21–23]. Oct4 regulates expression of several genes, including Fgf4, Rex1 (also known as Zpf42), Sox2, Ssp1 (osteopontin), Hand1 and Ifnt1 [23, 24]. The Oct4 promoter is active in oocytes, zygotes, early cleavage-stage embryos, the inner cell mass of the blastocyst and in embryonic carcinoma and stem cells [21, 24, 25]. Oct4 is downregulated when cells leave the germ line and is found exclusively in germ cells in fetal and adult mice [21–23]. (Re)activation of the Oct4 gene in somatic cells would be a strong indication that these cells fulfill an essential molecular precondition for pluripotency. To test the potential for multilineage differentiation and functional contribution to organogenesis, the most informative assay is injection of the putative stem cells into recipient blastocysts and the generation of chimeras.
Here, we report the identification of a fetal somatic stem cell (FSSC) population in primary fibroblast cultures derived from two mammalian species (mouse and pig) and show that simple changes of the culture conditions allow selective enrichment of the FSSCs. The FSSCs could be more amenable to reprogramming and would thus be a new source of donor cells in somatic nuclear transfer or cell-therapy approaches.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Primary cultures were prepared by somatic explant (<1 mm3) cultures of connective tissue. For isolation of fetal cells, porcine fetuses of Postcoitus (PC) Day 25 were eviscerated, the extremities and the heads were removed. Cell cultures from adult pigs were isolated from subdermal tissues of ear clips. The tissues were isolated under sterile conditions and washed twice in phosphate buffered saline (PBS) containing antibiotics and were then cut into small pieces and pasted to cell culture dishes by employing recalcified microdrops of bovine plasma. Bovine plasma was isolated from EDTA-blood of healthy cows by centrifugation (4000 x g, 15 min) and stored frozen in aliquots at –80°C. For use, an aliquot was thawed, supplemented with 80 mM CaCl2 and microdrops of 5–10 µl were pipetted into a cell culture dish. Small pieces of tissue were placed into the plasma drops, and culture dishes were incubated for 10–30 min at 37°C until plasma coagulation was completed. Subsequently, tissues were maintained in Dulbecco modified Eagles medium (DMEM) medium supplemented with 2 mM glutamine, 1% nonessential amino acids, 1% vitamin solution, 0.1 mM mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin (Sigma, Deisenhofen, Germany) containing 10% fetal calf serum (FCS; batch numbers 40G321K, 40G2810K; Gibco, Karlsruhe, Germany) and incubated in humidified 95% air with 5% CO2 at 37°C [16, 17]. Outgrowing cells were trypsinized and subpassaged once before cryopreservation. Typically, first outgrowing cells from fetal explants became visible within 24 h of incubation, and subpassaging was done after 7 days of culture. For high serum culture, the serum content of the standard medium was increased to 30% FCS. For suspension culture, colonies were selectively isolated and completely dissociated in a trypsin solution, then 104 cells were seeded into 35-mm bacteriological dishes. Every second day, 50% of the medium was replaced with new medium. To determine the maximal replicative limit, cultures were serially subpassaged and 12.5 x 103 cells were seeded per square centimeter in six-well dishes, trypsinized after 5–7 days, counted, and reseeded. The number of accumulated population doublings per passage was determined using the equation, PD = log(A/B)/log 2, in which A is the number of collected cells and B is the number of plated cells. Murine fibroblasts were obtained from Day 11.5– 15.5 fetuses or adult OG2 mice [14] (homozygous for the Oct4-GFP transgene) or from double transgenic fetuses of crosses of OG2 with Rosa26 mice. Confocal microscopy was applied to detect GFP using a Zeiss Axiomat LSM and an excitation wavelength of 488 nm. Embryonic stem (ES) cells (wildtype GS1 129/Sv) were cultured as described [26]. Animal handling was in accordance with institutional guidelines.
In some experiments, porcine FSSCs were cultured in standard medium (DMEM 10% FCS) supplemented with growth factors. As controls, high serum and standard cultures were run in parallel and, after 7 days, the three-dimensional-colony formation and alkaline phosphatase (AP) induction were determined. The following growth factors were supplemented: human leukemia inhibitory factor (LIF, 1000 U/ml; Chemicon International), epidermal growth factor (EGF, 0.1 ng/ml; TEBU GmbH, Offenbach, Germany), platelet-derived growth factor (PDGF, 1.0 ng/ml; TEBU GmbH), basic fibroblast growth factor (bFGF, 1.0 ng/ml; TEBU GmbH), endothelial cell growth factor (ECGF, 500 ng/ml; Roche Diagnostics, Mannheim, Germany), and insulin (250 ng/ml; Sigma).
In some experiments, porcine FSSCs were cultured in high serum medium supplemented with one of the following inhibitory peptides: 1) bFGF inhibitory peptide H-1948 (5 µM; Bachem, Weil am Rhein, Germany), 2) insulin-like growth factor (IGF-I) analog H-1356 (10 µM; Bachem), 3) PDGF antagonist H-8650 (10 µM; Bachem), and 4) insulin receptor (689-702) H-4212 (10 µM; Bachem). In parallel, high serum and standard cultures were run as controls and, after 7 days, three-dimensional-colony formation and AP induction were determined.
DNA contents were measured by fluorescent activated cell sorting as described [16]. In addition, nuclei from FSSCs were karyotyped and the ploidy status was assessed.
RT-PCR of Specific mRNAs
In brief, total RNA was isolated from cells grown in six-well dishes and 0.5 µg RNA was reverse transcribed (RT) into cDNA using random hexamers in a total volume of 40 µl. Aliqouts of 2.5 µl of the RT-reaction were used as templates for polymerase chain reaction (PCR). Murine Oct4 and Stat3 were amplified by PCR with the following primers and conditions: 5'-GGC GTT CTC TTT GGA AAG GTG TTC and 5'-CTC GAA CCA CAT CC TTC TCT (35 cycles, annealing temperature 57°C) for the murine Oct4; 5'-TCA AGC ACC TGA CCC TTA GG and 5'-CTG AAG CGC AGT AGG AAG GT (37 cycles, 58°C annealing temperature) for Stat3 (U06922). For the amplification of TNAP (AF285233), the following primer pair was used: 5'-ATG AGG GTA AGG CCA AGC AG and 5'-CCA CCA TGG AGA CAT TTT CC (34 cycles, 58°C annealing temperature). Porcine OCT4 was amplified with intron-spanning 5'-AGG TGT TCA GCC AAA CGA CC and 5'-TGA TCG TTT GCC CTT CTG GC primers (AJ251914) and 36 cycles. Glyceraldehyde phosphate dehydrogenase (GAPD) was amplified with the primer pair 5'-CCT CCA CTA CAT GGT CTA CAT GTT CCA GTA and 5'-GCC TGC TTC ACC ACC TTC TTG ATG TCA TCA (25 cycles, annealing temperature 60°C). The GAPD primers matched completely with the porcine ortholog (U48832) and showed a single nucleotide mismatch with the mouse ortholog (BC083065). The PCR reactions were performed in 20-µl volumes, consisting of 20 mM TrisHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphates, 1 µM of specific primer pairs and 0.5 U of Taq DNA polymerase (Gibco). Reactions without RT or without template were used as controls.
Measurement of Cell Proliferation by BrdU Incorporation
DNA synthesis was measured by 5-bromo-2'deoxy-uridine (BrdU) incorporation as described [17]. Incorporated BrdU was detected by a chromogenic immunoassay employing an anti-BrdU antibody conjugated with alkaline phosphatase.
Immunohistology
For antibody staining, cells were cultured on gelatinized coverslips. Sterile coverslips were placed in six-well cell culture dishes and covered with a 1% gelatine solution (in PBS) for 5 min. The gelatine solution was then removed and the semidry coverslips were seeded with cells. After 3– 4 days of culture, the cells were fixed in cold 80% methanol. The following monoclonal antibody dilutions were used: anti-vimentin (AMF-17b, 1: 200; Developmental Studies Hybridoma Bank, IA) and anticytokeratin (peptide17, 1:100; Sigma). A rhodamine-labeled secondary anti-mouse antibody (1:2000; Molecular Probes, Netherlands) was used. In some cases, the nuclei were counterstained with 1 mM Hoechst 33342 [27]. The samples were examined with an Olympus BX60 microscope equipped with phase-contrast and epifluorescence optics, using band-pass rhodamine and Hoechst filter sets.
Staining of Endogenous Alkaline Phosphatase Activity
Standard and high-density cell cultures were prepared in six-well cell cultures dishes. The cultures were washed with PBS, fixed in 3.7% paraformaldehyde for 15 min, washed in PBS, and then incubated in a solution containing 25 mM TrisHC (pH 9.0), 4 mM MgCl2, 0.4 mg sodium-
-naphtylphosphate, 1 mg/ml Fast Red TR (Sigma), and 0.05% Triton X-100 for 60 min.
Chimera Generation by FSSC Injection into Host Blastocysts
Rosa26 homozygous mice were obtained from Jackson Laboratory (Bar Harbor, ME) and mated with homozygous OG2 animals to generate double-transgenic fetuses carrying both marker genes, which were used to isolate FSSCs. Day 11.5—15.5 fetuses were isolated and employed for fetal cell cultures using the explant method described above.
For blastocyst injections, 6- to 10-wk-old female CD2F1 mice were superovulated with 10 U eCG at noon on Day –2, followed by 10 U hCG on Day 0, and were then mated with CD2F1 males. The next day, females were checked for plug formation. At Day 3.5, females were sacrificed and the uterine tracts were isolated and flushed with PBS containing 1% bovine serum albumin. Blastocysts were isolated and incubated in PBS/1% albumin at 37°C. Single blastocysts were transferred into a micromanipulation unit (Zeiss) and fixed with a holding pipette. On average, 2–15 double transgenic cells (OG2/Rosa26) were injected into the blastocoel with the aid of a microcapillary. In total, 8–10 blastocysts were transferred into the uteri of Day 2.5 or Day 3.5 pseudopregnant NMRI females that had been mated with vasectomized males. Fetuses were recovered at Day 10.5–15.5 and either stained for lacZ-positive cells [28] as whole mounts, or dissected and screened for GFP expression in genital ridges and other organs.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Fetal OG2-transgenic mice, which express the GFP gene under transcriptional control of the germ-line-specific Oct4 promoter, were employed for explant cultures. Explants of somatic tissue with an average size of <1 mm3 were isolated from fetuses at PC Days 11.5, 13.5, and 14.5; attached with microdrops of bovine plasma to cell culture dishes, and cultured in DMEM. Special care was taken to isolate explants from mesodermal tissue of the neck and shoulder regions. As expected, no GFP-positive cells were found in the tissue explants and in the first outgrowths after 2 days in culture (Fig. 1, C and D). However, after 8 days of culture, GFP-positive cells were detected within the outgrowing primary cells (Fig. 1, E and F), indicating (re)activation of the germ-line-specific Oct4-GFP marker cassette. As positive control, the genital ridges were isolated in parallel and primordial germ cells could be unequivocally detected by their strong expression of the Oct4-GFP marker (Fig. 1, A and B). Additional experiments suggested that the microenvironment of the plasma microdrops played an important role in stimulating the proliferation of GFP-positive cells (Fig. 1, G–I). Therefore, subpassages of the outgrowing cells were cultured in DMEM containing high fetal calf serum supplementation (30% FCS) and a population of 
10–3 GFP-positive cells (Fig. 1, G–I) could be maintained. Expression of the endogenous Oct4 gene was confirmed by RT-PCR (Fig. 1J). Oct4 is one of the best characterized genes for stemness. The presence of Oct4 transcripts has been shown by two independent assays in this study. The absence of a PCR product from fibroblast cultures argues against the amplification of an Oct4 pseudogene. In addition, the tissue nonspecific variant of alkaline phosphatase (TNAP) and Stat3 were expressed in high serum-supplemented cultures as determined by RT-PCR (Fig. 1); in contrast, no expression of the embryonic or intestinal AP genes could be detected (not shown).
|
In Vivo Differentiation Potential by Injection of FSSCs into Blastocysts
To determine the developmental potential, FSSCs were injected into murine blastocysts, which were subsequently transferred to pseudopregnant recipients. FSSCs of both sexes were isolated from double transgenic fetuses of OG2 and Rosa26 mouse strains. These cells carried the germline-specific Oct4 GFP and the ubiquitously active lacZ reporter gene constructs and thus allowed distinguishing them from the cells of the recipient blastocysts.
Day 10.5–15.5 fetuses derived from the injected blastocysts were isolated and analyzed for chimerism either by staining for lacZ activity or by fluorescence microscopy to identify GFP-positive cells. Of a total of 19 analyzed fetuses, 7 contained progeny cells from the injected FSSCs (Table 1). Chimerism was detected in mesenchymal organs, such as liver, muscle, and tongue, but also in the genital ridges. Figure 2A shows an example of a chimeric fetus with massive lacZ staining in liver, tongue, and genital ridges, suggesting that at least parts of these organs were derived from the injected cells. Chimeric and wildtype fetuses were derived from embryo transfers that had been performed on the same day, were stained for LacZ activity in parallel, and photographed on the same slide. It is unclear whether the apparent oversize of the chimeric fetus is related to the cell injection. The summarized data for the blastocyst transfer suggest that development of embryos after FSSC injection is compromised (Table 1). Figure 2B shows the presence of GFP-positive cells in the genital ridges of a male PC Day 15.5 fetus. In total, 16 GFP-positive cells were counted in the squeeze preparation, and these cells behaved like prospermatogonia in that they floated within the ducts of the genital ridges. GFP-positive cells were not found in other organs, such as heart, liver, brain, or connective tissue.
|
|
Isolation of FSSCs from Porcine Tissues
The isolation of FSSCs with stem-like properties from murine somatic explants raised the question whether this is a species-specific phenomenon or whether similar cells could be obtained from other species. Therefore, somatic explants of porcine fetuses (PC Day 25) were established and subpassaged once using standard protocols. Immunostaining showed uniform labeling for vimentin and no labeling for cytokeratin (data not shown), suggesting that most of the cells were fibroblasts. However, culture in DMEM with high serum supplementation activated the porcine OCT4 gene (Fig. 3M). Porcine OCT4 was amplified with intron spanning porcine-specific primers; the obtained PCR product was purified, sequenced, and found to be identical to the corresponding OCT4 cDNA sequence. Control cultures maintained in DMEM/10% FCS did not express detectable OCT4 mRNA levels
|
With high serum concentrations, the cultures grew faster and lost contact inhibition, resulting in the formation of three-dimensional colonies (Fig. 3, A and B). Only cells within the three-dimensional colonies continued to proliferate as measured by BrdU incorporation, whereas cells in the surrounding monolayers were mitotically inactive (Fig. 3D). Staining for endogenous AP activity revealed a massive induction of AP-positive cells, almost exclusively associated with the three-dimensional colonies (Fig. 3, G–J). AP-positive cells differed (Fig. 3, K and L) from typical fibroblasts in that they displayed dendritic projections. These changes were not found in control cultures from identical batches of cells when grown under standard conditions with 10% FCS supplementation (Fig. 3C, Table 2). In total, 6.7% of microwells seeded with 10 cells from high serum cultures resulted in continuously growing cultures, suggesting that 1 out of 150 cells was able to initiate clonal growth. Induction of colony growth and AP expression by high serum supplementation were abolished by pretreatment with heat (90°C) or trypsin (data not shown).
|
Adult porcine fibroblasts derived from subdermal tissue explants of three animals did not display three-dimensional-colony growth (Fig. 4) when cultured for 7 days in DMEM/ 30% FCS, but the frequency of AP-expressing cells increased 2- to 10-fold over this period (Table 2). In comparison, when fetal cells were used, a 100- to 1000-fold increase in AP-positive cells was observed. Induction of three-dimensional-colony growth and AP expression in porcine cultures was at least one order of magnitude higher than that seen in murine cultures (Table 2).
|
When high serum cultures were split and one part of the population was returned to standard medium, colony growth ceased and AP-positive cells disappeared in the standard culture within 2–3 subpassages, suggesting that the induction and proliferation of FSSCs are dependent on high levels of not-yet-identified serum factors (Fig. 4B).
When high serum cultures were subpassaged five times in standard medium, the colony growth phenotype was lost and a switch to high serum medium was no longer associated with the appearance of three-dimensional growth, suggesting that the FSSC population had been lost due to unfavorable conditions.
To narrow down the possibilities of potential factors in serum responsible for these effects, it was tested whether purified growth factors could substitute for high serum supplementation. Porcine FSSC cultures in standard medium (DMEM 10% FCS) were supplemented with either hLIF, PDGF, FGF1, ECGF, or insulin. However, none of these growth factors induced three-dimensional growth or induction of AP expression compared with approximately 200– 300 colonies in the positive controls (not shown).
In addition, no inhibitory effect of the inhibitory peptides FGF1 inhibitory peptide, IGF1 analog, PDGF antagonist, or insulin-receptor [609–702] [29–32] on the FSSC phenotype could be detected; the peptide supplemented cultures developed the same number of FSSC colonies as the positive controls. These data indicate that the examined growth factors and receptors are not involved in the apparent pluripotent phenotype induced by the high serum supplementation.
FSSCs Have Increased Proliferative Potential
Culture medium supplemented with high serum resulted in a dramatically altered growth curve (Fig. 4C). Porcine FSSC cultures maintained under high serum conditions grew continuously over a period of >120 days and exceeded more than 100 cell doublings without reaching a plateau phase (Fig. 4C). In contrast, standard cultures, i.e., DMEM with 10% FCS, were compatible with only 50–60 cell doublings before mitotic activity ceased after approximately 70 days. The FSSCs maintained a diploid status, as measured by fluorescence-activated cell sorting (Fig. 4D) and metaphase spreads of cells from passage 20. Thirty-two karyotyped nuclei of porcine FSSC cultures contained the euploid number of 38 chromosomes; only 1 nucleus with 37 chromosomes was found.
FSSCs Are Capable of Forming Spheroids and Exhibit Anchorage-Independent Growth
To investigate the growth potential of the colony-forming cells, porcine three-dimensional colonies of 200–300 µm diameter were isolated and trypsinized to obtain single-cell suspensions. Subsequently, 104 cells were seeded into bacteriological culture dishes. In DMEM/30% FCS, irregular aggregates consisting of only few cells (2–20), were detected. These cells did not divide and the majority apparently underwent cell death (Fig. 4E). Culture medium supplemented with retinoic acid induced the formation of tiny aggregates, which, after 2–4 days, attached to the surface (Fig. 4F). Supplementation of the culture medium (DMEM/30% FCS) with dexamethasone resulted in aggregation of small multicellular spheroids within 24 h, which continued to grow up to a diameter of >400 µm after 10– 15 days and contained nearly exclusively AP-positive cells (Fig. 4, G and H). Dexamethasone is an antiinflammatory glucocorticoid, which regulates T cell survival, growth, and differentiation and inhibits the induction of nitric oxide synthase. Several protocols for differentiation of stem cells also employed dexamethasone. The dexamethasone-derived spheroids were capable of forming secondary spheroids if trypsinized and reseeded in bacteriological dishes. If plated on gelatinized coverslips, dexamethasone spheroids reattached and monolayer cells grew out. These outgrowing cells were vimentin negative, whereas control cultures kept in standard medium with 10% FCS were strongly positive (Fig. 4, I and J).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chimerism was found preferentially in liver, muscle, and tongue. The LacZ-positive cells in the tongue may actually represent lymphatic tissue. It is well known that the fetal liver contains hepatic as well as migratory hematopoietic progenitor cells. However, we have not yet determined which cell types in these three organs are derived from the injected cells. This is the subject of future studies. No chimerism was detected in heart and brain, two organs that showed a high rate of spontaneous cell fusions in a recent study [12]. However, we cannot fully exclude the possibility that fusion with differentiated cells might have contributed to the observed chimerism.
FSSCs represent a rare subpopulation in fetal fibroblast cultures, and the proliferation of FSSCs critically depends on the culture conditions, including an autologous feeder-cell layer and factors present in bovine serum. The explant culture is based on microdrops of bovine plasma as an adhesive support, which seems to be essential for the initial stimulation of FSSC proliferation. Standard culture media compositions, with no or low amounts of FCS supplementation, are associated with a progressive loss of the FSSC phenotype. These observations show that appearance of FSSCs is critically dependent on cell-collection method and/or culture conditions.
At present, it is not clear whether FSSCs represent a preexisting subpopulation in the fetus or whether they are the result of a de novo formation induced by the culture conditions. Potential sources are ectopic primordial germ cells [19] or fetal tissue-specific stem cells. In humans, it has been shown that mesenchymal stem cells are present in a variety of tissues during development, whereas in adults, they are predominantly found in the bone marrow [33]. Activation of the Oct4 promoter in murine explant outgrowths, although at lower expression levels than in murine ES cells, after a few days in culture suggests that the culture conditions induce this phenotype. We cannot completely rule out that this reprogramming is attributed to dedifferentiation of a susceptible, non-lineage-committed cell population present in fetal tissues. The observed frequency of 1/1000 GFP-positive cells in the in vitro cultures may actually underestimate the occurrence of FSSCs, as GFP expression was low and might be around the detection limit. Clonal growth was found for 1 out of 150 cells and chimerism was detected in 7 out of 19 fetuses.
In line with the expression of stem cell marker genes, FSSCs show three-dimensional-colony growth in adherent culture and spheroid growth in suspension culture, whereas fibroblasts cease dividing and arrest in the G1 phase of the cell cycle under these conditions [34–36]. Loss of contact-inhibition and anchorage-independent growth is characteristic for ES cells or transformed cells [37]. Our data provide convincing evidence that, unlike many cell lines derived from tumors or cells transformed by oncogenic agents, the FSSC subpopulation does not result from spontaneous immortalization or transformation. FSSCs do not exhibit a crisis followed by clonal outgrowth, and they show no chromosomal abnormalities or aneuplodies. The altered growth characteristics of FSSCs are reversed after exposure to standard cell culture conditions. Unlike rodent cell cultures, cells from humans and livestock rarely, if ever, immortalize spontaneously [38]. Our findings show that FSSCs can be efficiently and reliably isolated from porcine, but also from bovine and ovine, fetal explants (data not shown) and with a lower efficiency from murine fetal explants. The diminished efficiency of FSSC stimulation from murine explant cultures may be attributed to the species specificity of factors present in bovine serum.
In conclusion, this study shows that explants from fetal somatic tissues contain a subpopulation of potentially pluripotent stem cells, which fulfill most of the essential criteria for stem cells, including self-renewal ability, multilineage differentiation, and differentiation in vivo [39]. Therefore, the FSSCs might hold great promise for regenerative therapies.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: FAX: 0 5034 871 101; niemann{at}tzv.fal.de
Received: 26 April 2004.
First decision: 11 May 2004.
Accepted: 9 December 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Goel, M. Fujihara, N. Minami, M. Yamada, and H. Imai Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis Reproduction, June 1, 2008; 135(6): 785 - 795. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. K Berg, C. Li, G. Asher, D. N Wells, and B. Oback Red Deer Cloned from Antler Stem Cells and Their Differentiated Progeny Biol Reprod, September 1, 2007; 77(3): 384 - 394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Piliszek, J. A Modlinski, K. Pysniak, and J. Karasiewicz Foetal fibroblasts introduced to cleaving mouse embryos contribute to full-term development Reproduction, January 1, 2007; 133(1): 207 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. A. Kues, R. Schwinzer, D. Wirth, E. Verhoeyen, E. Lemme, D. Herrmann, B. Barg-Kues, H. Hauser, K. Wonigeit, and H. Niemann Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs FASEB J, June 1, 2006; 20(8): 1200 - 1202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Zipori The Stem State: Plasticity Is Essential, Whereas Self-Renewal and Hierarchy Are Optional Stem Cells, June 1, 2005; 23(6): 719 - 726. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) containing 10% FCS and high serum medium (
) containing 30% FCS. D) Cell cycle status in standard and high serum culture. Note that the high serum culture displays a normal ploidy. E, F) Anchorage-independent growth of FSSCs in suspension culture. High serum-induced three-dimensional colonies were isolated, trypsinized to single cell suspensions, and seeded into bacteriological dishes to prevent attachment. E) In DMEM with high serum supplementation, tiny aggregates formed. F) In high serum medium with retinoic acid (10–7 M), the initial aggregates reattach and show outgrowing cells on the surface. G) In high serum medium with dexamethasone (10–7M), spheroids of >300-µm size grow over 10–15 days; inset: lower magnification. H) Dexamethasone-spheroids stained for endogenous AP, bar = 230 µm. I) Expression of vimentin in fibroblasts cultured in standard medium (passage 5), merged image of antibody (red) and nuclei (blue) staining (left). J) Loss of vimentin epitopes in cells derived from dexamethasone spheroids. After 15 days of suspension culture, the spheroids were allowed to reattach to gelatinized coverslips and probed with a monoclonal antivimentin antibody