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The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Key Words. Embryonic stem cells • Mesenchymal stem cells • Marrow stromal (stem) cells • Self-renewal • SSEA-4 • Oct-4
Correspondence:
Linzhao Cheng, Ph.D., The Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer Research Building, Room 208, 1650 Orleans Street, Baltimore, Maryland 21231, USA. Phone: 410-614-6958; Fax: 410-614-7279; e-mail: lcheng{at}welch.jhu.edu
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ABSTRACT |
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INTRODUCTION |
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In adults, several distinct types of multipotent stem cells have been isolated from bone marrow (BM). Adult BM is the primary site of hematopoietic stem cells (HSCs), the common precursor of blood and immune cells. Beginning with pioneering work by Friedenstein et al. more than 30 years ago, it is well recognized that non-HSCs are also present in the BM of adult humans and animals [8,9]. The most characterized type is mesenchymal stem cells capable of generating mesenchymal cells and stromal cells that support hematopoiesis [10–15]. It was also reported in recent years that certain freshly isolated or culture-expanded BM cells can differentiate into many other types of cells such as hepatocytes in the liver, neurons and glial cells in the brain, satellite cells in skeletal muscles, and cardiomyocytes in the heart [16–24]. Thus, adult BM contains cells and microenvironments that are able to maintain stem cells in their undifferentiated states. These studies led us to investigate whether adult BM-derived cells can also support the growth of hES cells.
In recent years, several groups have developed improved methods to obtain large numbers of marrow stromal progenitor cells in culture from adult human BM aspirates, either by physical enrichment of precursor cells followed by culture expansion, or by direct culture selection and amplification. These marrow fibroblastic cells have been termed as either stromal progenitor cells reflecting their proliferation potential in culture [11], marrow stromal cells reflecting the source and method of the derivation [12–14], or mesenchymal stem cells reflecting their proven potentials to generate multiple types of mesenchymal cells when exposed to appropriate stimuli in vivo or in vitro [10,25]. The latter two methods, which are very similar in practice, are widely used by many investigators including ourselves [26]. These marrow-derived (fibroblastic) stromal cells that function as nonhematopoietic multipotent stem cells are collectively called herein as MSCs. After two passages (approximately 14 cell divisions) in a selective medium supplemented with fetal bovine serum (FBS), culture-expanded human MSCs (hMSCs) are morphologically and phenotypically homogenous and essentially free of endothelial cells and macrophage or adipocyte contamination [25,26].
These culture-expanded and highly homogenous hMSCs enable us to perform detailed analyses that were previously impossible with mixed stromal cell populations. When used as adherent feeder cells with culture media optimized for hematopoietic cells, the culture-expanded hMSCs supported human CD34+ HSCs in long-term culture assays and their differentiation into erythroid, myeloid, megakaryocytic, osteoclastic, or B-cell lineages even in the absence of added cytokines [26–31]. The activity is due to, at least in part, the production of various hematopoietic cytokines, including LIF, interleukin (IL)-6 and IL-11, as well as stem cell factor and Flt3/Flk2 ligand by hMSCs [26–28,31]. Thus, our goal of the present study was to investigate whether culture-expanded hMSCs derived from human adult BM can also support the growth of hES cells in a culture medium that is known to favor their proliferation while retaining the undifferentiated state.
We report here that culture-expanded MSCs can replace MEFs and fully support prolonged expansion of hES cells in culture. Human ES cells cocultured on irradiated hMSCs expanded >100-fold during the 30-day continuous culture (in five passages). The expanded hES cells after nine passages maintained their normal karyotype. Moreover, the expanded hES cells retained the unique hES cell morphology and expression of APase and SSEA-4 that are characteristic of undifferentiated hES cells.
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MATERIALS AND METHODS |
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Human ES Cells
The H1 (also known as WA01 in the National Institutes of Health Embryonic Stem Cell Registry) hES cell line (p22) was obtained from the WiCell Research Institute (Madison, WI; http://www.wicell.org) and initially cultured on MEFs as instructed by the provider. MEFs (p3) purchased from Specialty Media, Inc. (Phillipsburg, NJ; http://www.specialtymedia.com) were used as feeder cells for the hES cells. MEFs or MSCs (200,000 cells) were plated per well (9.4 cm2) in 6-well plates after being irradiated (50 Gy, 1 Gy = 100 rads) using a 137Cs gamma-irradiator. The hES cell culture medium consisted of 80% (v/v) knockout (KO) DMEM, 20% (v/v) of the KO serum replacement, 2 mM L-glutamine, 10 mM nonessential amino acids (all from Invitrogen), 50 µM ß-mercaptoethanol (Sigma), and 4 ng/ml bFGF. Cell cultures were incubated at 37°C in 5% CO2 in air and 95% humidity. Once hES cell colonies grew to maximal size before the onset of visible differentiation, cells in the coculture (hES cells and irradiated MEFs) were digested and seeded onto newly prepared feeder cells. Initially, we used collagenase IV (1 mg/ml; Invitrogen) to split cells (1:1 to 1:3) as instructed and published previously [1,3]. Later, cells were harvested by digesting cells in cocultures with 0.05% trypsin/0.53 mM EDTA solution for 5 minutes. The digestion was stopped by adding soybean trypsin inhibitor (0.5 mg/ml; Sigma). After washing, the dissociated cells in the hES cell culture medium were split from 1:1 to 1:50 and seeded onto feeder cells or 6-well plates coated with diluted (1:20) Matrigel (Becton Dickinson Labware; Bedford, MA; http://www.bd.com) as described previously [7].
Immunofluorescence and APase Staining
Cocultures used for APase staining or immunofluorescence analysis were established in either 6-well or 24-well plates. Prior to analysis, adherent cell layers were fixed by the addition of 10% formalin (15 minutes). After wash with a Tris-based saline solution, APase staining was performed using a kit containing BCIP/NBT as the substrate (Sigma). The dark blue staining was visualized by light microscopy. The fixed cells in cocultures were also stained with mouse mAbs against either SSEA-4 (clone MC-813-70, isotype IgG3) or SSEA-1 (clone MC-480, isotype IgM). Hybridoma supernatants of both mAbs were obtained from Developmental Studies Hybridoma Bank (Iowa City, IA; http://www.uiowa.edu/
dshbwww). For the immunofluorescence staining, the fixed cells were incubated for 15 minutes with goat serum (2%) to block nonspecific binding. The cocultures were stained with diluted (1:100) hybridoma supernatants recognizing either SSEA-4 or SSEA-1 antigen. After incubation in the dark for 1 hour at 25°C or overnight at 4°C, fixed cells were washed extensively before the secondary staining reagent was added. Goat anti-mouse IgG conjugated to the fluorochrome Alexa 546 (Molecular Probes; Portland, OR; http://www.probes.com) was added for 45 minutes at 25°C. The nuclei of hES cells and hMSCs were counterstained by Hoechst 33358 (Molecular Probes). Immunofluorescence analysis was performed with a Nikon (TE300) microscope with separate filters for either Hoechst (blue) or red fluorescence, or a triple filter for blue, green, and red fluorescence simultaneously. The fluorescence and light images were recorded on Kodak film (ASA400). The scanned image was analyzed by Photoshop 4.0.
Cell Isolation by Magnetic Cell Sorting (MACS)
Cells were harvested from cocultures by gentle digestion with 0.25% trypsin/0.53 mM EDTA solution and washed once with PBS containing 2% bovine serum antigen (BSA) and 2 mM EDTA. Before incubating with the SSEA-4 mAb (1:100), cells were preincubated with human IgG (2 mg/ml) to block nonspecific IgG binding. The SSEA-4-labeled cells were incubated with magnetic beads conjugated with anti-mouse IgG antibodies (Miltenyi Biotec; Auburn, CA; http://www.miltenyibiotec.com). The labeled cells were isolated by the miniMACS magnet stand and the large cell isolation column as instructed (Miltenyi Biotec).
Flow Cytometric Analysis
Cells were harvested as described above and suspended in 100 µl staining buffer (2% BSA, 2 mM EDTA, and 0.1% sodium azide in PBS) containing human IgG to block nonspecific IgG binding. The diluted (1:100) SSEA-1 or SSEA-4 mAbs were added as primary antibodies. Fluorescein isothiocyanate (FITC)- or R-phycoerythrin (PE)-conjugated anti-mouse IgG antibodies were used to detect the binding of SSEA-4 mAb (mouse IgG3). For the SSEA-1 mAb (mouse IgM), anti-mouse IgM antibodies conjugated with PE were used. These secondary reagents were purchased from Caltag (Burlingame, CA; http://www.caltag.com) or Pharmingen (San Jose, CA; http://www.bdbiosciences.com/pharmingen). In addition, we also used PE-conjugated mAbs recognizing SH-2/endoglin/CD105 (clone SN6 or 266), HLA-ABC/ major histocompatibility complex (MHC) class I (clone TU149), HLA-DR/MHC class II (clone L233), CD133 (clone AC133-1), Thy-1/CD90 (clone F15-42-1-5), CD34 (clone HPCA-2), and platelet endothelial cell adhesion molecules (PECAM)-1/CD31 (MBC78.2), in conjunction with SSEA-4 and the FITC-conjugated anti-mouse IgG antibody (for hES cells). These PE-conjugated mAbs were purchased from Caltag, Miltenyi Biotec, Beckman Coulter (Miami, FL; http://www.beckman.com), and Pharmingen, and used as instructed by providers. A FACScan® flow cytometer (Becton Dickinson) was used for these analyses. Ten thousand events were acquired for each sample and analyzed using CellQuest software (Becton Dickinson).
Embryoid Body (EB) Formation and Differentiation In Vitro
Prior to differentiation, hES cells were collected and resuspended in the hES cell medium in the absence of bFGF. The hES cells were cultured in 6-well plates as aggregates in suspension. After 5 days, FBS (5% final) was added. Typically, aggregated hES cells formed EBs after 7-10 days in suspension culture, and mature (cystic) EBs emerged subsequently from 20%-80% of formed EBs.
All-trans-retinoic acid (RA) was purchased from Sigma and dissolved in dimethyl sulfoxide to make a 10-2 M stock solution. The RA induction of hES cell differentiation was carried out essentially the same as described previously [5]. Briefly, hES cells that had been cultured on hMSCs for eight passages were passaged onto Matrigel in the hES cell medium containing bFGF. RA (10-5 M final) was added the next day into half of samples. The culture medium was exchanged every 2 days. At day 9, cells were fixed and stained with APase, SSEA-4, and Hoechst dye as described above.
Karyotypic Analyses of hES Cells
Before and after cocultures on hMSCs, karyotyping analyses of hES cells were carried out by the Laboratory of Prenatal and Research Cytogenetics in the Department of Obstetrics and Gynecology at Johns Hopkins Hospital. The method used is essentially the same as previously described [32]. Briefly, cells were incubated with 0.1 µg/ml of colcemid for 3-4 hours, trypsinized, resuspended in 0.075 M KCl, incubated for 20 minutes at 37°C, and then fixed in 3:1 methanol/acetic acid. After staining, karyotypes of normal human chromosomes were examined by cytogenetics specialists at the 300-band level of resolution. For hES cells (the H1 line is from a male embryo) that had been cultured with male hMSCs (irradiated) for seven passages, we first passaged the coculture onto female hMSC feeder cells for two more passages followed by two additional passages on MEFs. Male hES cells could then be easily identified from irradiated mouse cells or fewer irradiated female hMSCs.
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RNA Preparation and Gene Expression Analysis |
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RESULTS |
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Improved Method to Derive and Expand hMSCs
The method used to derive and expand hMSCs from adult BM is similar to that previously described [25,26] with a few modifications. A major change is to add bFGF (1 ng/ml) to the culture medium containing 10% FBS. Consistent with a previous report [34], we found that adding a low concentration of bFGF provides a consistently optimal growth condition and essentially alleviates the need to screen favorable FBS lots. Using this improved method, we can efficiently and consistently derive and expand hMSCs from multiple male and female donors. After the primary and two subsequent passages in culture (a total of ~6 weeks), 75-200 million (p2) hMSCs can be obtained from ~100 x 106 MNCs in a 10-cc BM aspirate sample. Similar to the previous method, the expanded hMSCs were highly uniform in morphology and phenotype and essentially free of adipocytes, hematopoietic cells (CD45+), and endothelial cells (CD34+ or CD31+). As previously described [25,31], the expanded hMSCs in culture expressed unique markers such as CD105 (also known as SH-2 and endoglin) and CD90/Thy-1 (Table 1
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. In the absence of seeded hES cells (i.e., hMSCs alone), no hES cell-like colonies formed with or without irradiation (not shown). On nonirradiated hMSCs, hES cell colonies had a better growth rate and more compact morphology (Fig. 1A). However, the proliferation of hMSCs in coculture imposed a practical difficulty, i.e., overgrowth of hMSC feeder cells that divided faster than hES cells. Subsequently, we discontinued the coculture of hES cells with proliferating MSCs and used exclusively irradiated MSCs as feeder cells. On irradiated hMSCs, hES cell colonies amplified >100-fold during the 30-day continuous culture (in five passages). In multiple experiments performed to date, the longest continuous coculture of hES cells with irradiated MSCs is 13 passages before the expanded hES cells were used for various assays.
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). Both MSC feeders gave rise to similar numbers of hES cell colonies. We estimated that there was at least fivefold expansion in this passage. So far, different preparations (p2 to p5) of hMSCs from three donors (two males and one female) have been used, all with similar results (not shown). Under the same culture condition, Matrigel was insufficient to support hES cell growth, regardless of whether the conditioned medium from MSCs or MEFs was provided (not shown). The MEF feeder gave rise to fewer and smaller ES cell colonies (~twofold expansion). Therefore, we found that hES cells appeared to grow better on hMSCs than MEFs once they adapted on hMSCs (six passages in this experiment). This result suggests an "adaptation" hypothesis, which also explains the fact that hES cells grew poorer initially on hMSCs when passaged initially from the coculture with MEFs.
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). APase activities were preferentially expressed on the cell membrane of hES cells and sensitive to levamisole inhibition (not shown). Like irradiated MEFs, few singular MSCs with apoptotic morphology (broad and flat) displayed a weak APase activity (not shown). The absence or low level of APase activities on the viable MSCs is consistent with the report that undifferentiated hMSCs are APase negative until induced to differentiate to osteoblasts [35]. The expanded hES cells were also stained for SSEA-4, a glycolipid antigen expressed on hES cells but not on mouse ES cells. After fixation, the cocultures of hES cells with irradiated hMSCs (p4) were stained with or without a mouse mAb against the SSEA-4 antigen (Fig. 3). A high level of SSEA-4 expression was found in expanded hES cells but was absent in the hMSC feeder cells (Fig. 3A and 3C).
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). Flow cytometric analysis confirmed that the hES cell fraction (8.6% in Fig. 4A) retained a high level of the SSEA-4 expression. After the MACS isolation, the purity of hES cells (SSEA-4+) was 93%-97% (94% in Fig. 4B). The isolation method allowed us to obtain a pure hES cell population essentially free of feeder cells.
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), we analyzed the gene expression of Oct-4, a critical transcription factor that is known to be preferentially expressed in undifferentiated hES cells [1–3]. By RT-PCR (Fig. 5), a high level of the Oct-4 expression was detected in the hES cells cultured on MEFs (lane 1) as described previously [1–5]. We also found that hES cells that had been cultured on hMSCs for five passages also expressed the Oct-4 gene at a high level (lane 3). In contrast, hMSCs alone expressed a very low but detectable level of the Oct-4 gene (lane 4). The authenticity of the Oct-4 RT-PCR product was confirmed by DNA sequencing (not shown).
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. The results are summarized in Table 1. The expression pattern of these unique yet not definite markers is consistent with previous reports with H1 and several other hES cell lines [5,33,36]. Based on their morphology, the Oct-4 gene RT-PCR analysis, and the expression of 11 unique cell-surface markers including APase and SSEA-4, we conclude that the hES cells expanded by coculture on MSCs retained a unique morphology and phenotype characteristic of undifferentiated hES cells, as they did on MEFs.
Expanded hES Cells on MSCs Retain Unique Potential of Differentiation in Culture
We used aliquots of the expanded hES cells that had been cultured on hMSC for 8-12 passages in two culture assays to test whether they retained unique differentiation potentials. One such assay is to form EBs in suspension, a complex and organized structure resembling early embryos [2–5]. Figure 6A shows a representative EB derived from aggregated hES cells that had been cultured on hMSCs for 12 passages and then in suspension culture for 15 days. When placed on tissue culture grade plates in FBS-containing medium, EBs adhered to the substrate and became flat cell masses that quickly proliferated and outgrew. A snapshot of outgrown differentiated cells 7 days in culture, which had morphology distinct from the undifferentiated ES cells, is shown in Figure 6B. When hematopoietic cytokines were added into the differentiation medium, hematopoietic-like cells were observed (Fig. 6C).
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). The RA-treated cells (Fig. 7A) showed very different morphology (colonies with discrete cells) compared with the untreated hES cells (Fig. 7B, more compact colonies). It is more evident after cell nuclei staining (Fig. 7C and 7D). The RA-treated cells displayed a lower level of APase or SSEA-4 after the 9-day treatment, regardless of whether they had been originally cultured on hMSCs or MEFs (data not shown). Also consistent with the previous report [5], we found that hES cells appear to be less sensitive to the RA induction (or slower) as compared with mouse ES cells. Together with the previous data showing the undifferentiated morphology and phenotypes (Figs. 3–5), the two in vitro differentiation assays further confirmed differentiation potentials of the expanded hES cells on hMSCs.
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shows a representative readout of such chromosomal karyotyping. Of the five samples examined, all of them displayed the same normal 44+XY chromosomal karyotype as the original. Thus, hES cells retained a stable and normal karyotype after prolonged expansion on hMSCs.
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DISCUSSION |
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Currently, we do not know the identity of molecules that are made by hMSCs or MEFs and required by hES cells in culture. In Figure 2, we showed that Matrigel alone (but with the conditioned medium from hMSCs or MEFs) was insufficient to support the growth of hES cells. Our result and a recent report [37] contradicted a previous report by Xu et al. [7] that hES cells could be maintained in an undifferentiated state if they were cultured on Matrigel as the ECM together with (undiluted) conditioned medium from MEFs. The apparent discrepancy may be due to varied levels of "carryover" MEFs in the Matrigel feeder cell-free culture. Xu et al. did not use purified hES cells free of MEFs that are also present in cocultures and did not address the level and importance of the carryover MEFs in their Matrigel feeder cell-free culture [7]. Since the majority of irradiated MEFs in hES cell coculture survived for at least 2 weeks, the carryover MEFs from the previous passage, although reduced in numbers, were also present in the Matrigel culture for several passages. For example, when an aliquot of hES cell cocultures (e.g., 1:3) was plated on Matrigel-coated plates, the equal fraction of MEFs (one-third of 200,000 cells) was also seeded simultaneously in the new culture.
Using the method described by Xu et al. [7], indeed we could also maintain or moderately (~twofold) expand hES cell colonies in the subsequent passage, if the splitting ratio of cocultures was between 1:1 and 1:3. However, we were unable to maintain the majority of hES cells in an undifferentiated state on Matrigel for four or five passages (~30-40 days, when numbers of remaining MEFs were negligible) and no expansion of ES cell colonies was observed after 1 month. Similarly, we failed to maintain (no net reduction in numbers of) undifferentiated hES cell colonies on Matrigel after one passage, if purified hES cells or significantly diluted cocultures (5% or 1:20, so that the carryover MEFs were negligible) were seeded (Fig. 2). Our data are consistent with a recent publication reporting the similar difficulty in passaging and maintaining hES cell lines in an undifferentiated state using Matrigel supplemented with conditioned medium for >35-42 days [37]. Our data together with Richards et al. [37] acknowledged our current inability to separate the soluble and cell-associated components required for prolonged growth and expansion of hES cells in culture.
The culture-expanded and highly homogenous hMSCs will enable us to perform detailed analyses, which were previously impossible with mixed cell populations such as poorly characterized (p3) MEFs. A number of reports examined the production of cell adhesion molecules and growth factors/cytokines by hMSCs. For example, we have detected mRNA and protein for LIF, as well as IL-6 and IL-11 [27]. Indeed, hMSCs fully supported the proliferation of undifferentiated mouse ES cells in the absence of exogenous LIF, either in FBS-containing or the hES cell culture medium (unpublished data, August 2002). To this end, we will continue the investigation to separate soluble factors and ECM/cell adhesion molecules made by hMSCs. It is of interest to determine whether human feeder cells produce these required proteins only after they are cultured in the hES cell culture medium. It remains to be determined whether the ability of hMSCs to support the prolonged growth of hES cells is dependent on the MSC potential to generate various mesenchymal cell lineages.
Richards et al. also reported that fetal skin and muscle cells from 14-week-aborted fetuses can also support prolonged growth of hES cells [37]. Although it is an important breakthrough, "ethical concerns regarding the derivation of fetal cells from human abortuses" limit their uses, as noted by the authors [37]. They also used human feeder cells derived from adult fallopian tubal (AFT) tissues after hysterectomy. Unless the derived AFT cells can be immortalized and proliferate indefinitely in culture, the use of primary AFT cells to culture various ES cell lines for ES cell expansion will not be practical. In comparison, hMSCs can be readily derived from adult healthy donors or prospective patients, and can be expanded millionfold before being used in coculture for supporting hES cell expansion. Once hMSCs prove to support other ES cell lines and/or the derivation of new ES cell lines, this novel serum-free, animal cell-free culture system will provide a clinically and ethically feasible method to vastly expand hES cells on a clinical scale. Currently, hMSCs are established and expanded with media containing 10% FBS. Ideally, we will find a way to eliminate or replace FBS with human serum or purified factors.
We should also point out that hMSCs cultured with FBS have been used in several clinical trials for autologous and unrelated (allogeneic) transplantation and no adverse effects associated with the use of FBS have been reported [31,38]. An additional advantage of using hMSCs to expand hES cells is that unrelated MSCs do not generate alloreactive T lymphocytes in culture or in large animals [31,38]. In fact, recent data even revealed the remarkable ability of hMSCs to downregulate host alloimmune responses to the third party graft [38,39]. One can envision that the presence of hMSCs (derived from the patient or from a universal source) may help to induce immune tolerance and reduce the allogeneic rejection to the hES cell-derived progeny (a third party graft), when transplanted into a patient whose genotype is different from the hES cells.
Although hMSCs can proliferate in culture for a long time, we often observed that their proliferation rate was significantly reduced after six passages (>25 population doublings). Three groups recently reported on the immortalization of the proliferative and primitive potentials of hMSCs by overexpressing the TERT gene, the catalytic subunit of telomerase [40–42]. Moreover, Dr. Verfaillie’s group reported that the adult BM-derived multipotent adult progenitor cells proliferate in culture for an extended time while maintaining their pluripotent differentiation potentials [21–24]. It is of great interest to determine whether these immortalized adult marrow-derived cells can also support hES cell expansion as did the hMSCs used in this report.
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ACKNOWLEDGMENT |
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REFERENCES |
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| EDITOR’S NOTE For another important paper on human ES see:
"High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors," by
Yue Ma, Ali Ramezani, Rachel Lewis, Robert G. Hawley, and James A. Thomson. STEM CELLS 2003;21:111–117
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|
|
|
|
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|
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|
|
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|
|
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G. Dravid, Z. Ye, H. Hammond, G. Chen, A. Pyle, P. Donovan, X. Yu, and L. Cheng Defining the Role of Wnt/{beta}-Catenin Signaling in the Survival, Proliferation, and Self-Renewal of Human Embryonic Stem Cells Stem Cells, October 1, 2005; 23(10): 1489 - 1501. [Abstract] [Full Text] [PDF]
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T. Miki, T. Lehmann, H. Cai, D. B. Stolz, and S. C. Strom Stem Cell Characteristics of Amniotic Epithelial Cells Stem Cells, October 1, 2005; 23(10): 1549 - 1559. [Abstract] [Full Text] [PDF]
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A. Yanagisawa, C. Endo, K. Okawa, S. Shitara, H. Kugoh, M. Kakitani, M. Oshimura, and K. Tomizuka Generation of Chromosome-Specific Monoclonal Antibodies Using In Vitro-Differentiated Transchromosomic Mouse Embryonic Stem Cells Stem Cells, October 1, 2005; 23(10): 1479 - 1488. [Abstract] [Full Text] [PDF]
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T. Li, S. Wang, Y. Xie, Y. Lu, X. Zhang, L. Wang, S. Yang, D. Wolf, Q. Zhou, and W. Ji Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monkey Embryonic Stem Cells Stem Cells, September 1, 2005; 23(8): 1192 - 1199. [Abstract] [Full Text] [PDF]
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Q. Wang, Z. F. Fang, F. Jin, Y. Lu, H. Gai, and H. Z. Sheng Derivation and Growing Human Embryonic Stem Cells on Feeders Derived from Themselves Stem Cells, September 1, 2005; 23(9): 1221 - 1227. [Abstract] [Full Text] [PDF]
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P. Stojkovic, M. Lako, S. Przyborski, R. Stewart, L. Armstrong, J. Evans, X. Zhang, and M. Stojkovic Human-Serum Matrix Supports Undifferentiated Growth of Human Embryonic Stem Cells Stem Cells, August 1, 2005; 23(7): 895 - 902. [Abstract] [Full Text] [PDF]
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 T. Fujikawa, S.-H. Oh, L. Pi, H. M. Hatch, T. Shupe, and B. E. Petersen Teratoma Formation Leads to Failure of Treatment for Type I Diabetes Using Embryonic Stem Cell-Derived Insulin-Producing Cells Am. J. Pathol., June 1, 2005; 166(6): 1781 - 1791. [Abstract] [Full Text] [PDF]
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P. Stojkovic, M. Lako, R. Stewart, S. Przyborski, L. Armstrong, J. Evans, A. Murdoch, T. Strachan, and M. Stojkovic An Autogeneic Feeder Cell System That Efficiently Supports Growth of Undifferentiated Human Embryonic Stem Cells Stem Cells, March 1, 2005; 23(3): 306 - 314. [Abstract] [Full Text] [PDF]
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S. Aggarwal and M. F. Pittenger Human mesenchymal stem cells modulate allogeneic immune cell responses Blood, February 15, 2005; 105(4): 1815 - 1822. [Abstract] [Full Text] [PDF]
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J. B. Lee, J. E. Lee, J. H. Park, S. J. Kim, M. K. Kim, S. I. Roh, and H. S. Yoon Establishment and Maintenance of Human Embryonic Stem Cell Lines on Human Feeder Cells Derived from Uterine Endometrium under Serum-Free Condition Biol Reprod, January 1, 2005; 72(1): 42 - 49. [Abstract] [Full Text] [PDF]
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J. B. Lee, J. M. Song, J. E. Lee, J. H. Park, S. J. Kim, S. M. Kang, J. N. Kwon, M. K. Kim, S. I. Roh, and H. S. Yoon Available human feeder cells for the maintenance of human embryonic stem cells Reproduction, December 1, 2004; 128(6): 727 - 735. [Abstract] [Full Text] [PDF]
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T. C. Schulz, S. A. Noggle, G. M. Palmarini, D. A. Weiler, I. G. Lyons, K. A. Pensa, A. C.B. Meedeniya, B. P. Davidson, N. A. Lambert, and B. G. Condie Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum-Free Suspension Culture Stem Cells, December 1, 2004; 22(7): 1218 - 1238. [Abstract] [Full Text] [PDF]
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C. Xu, J. Jiang, V. Sottile, J. McWhir, J. Lebkowski, and M. K. Carpenter Immortalized Fibroblast-Like Cells Derived from Human Embryonic Stem Cells Support Undifferentiated Cell Growth Stem Cells, November 1, 2004; 22(6): 972 - 980. [Abstract] [Full Text] [PDF]
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M. Stojkovic, M. Lako, T. Strachan, and A. Murdoch Derivation, growth and applications of human embryonic stem cells Reproduction, September 1, 2004; 128(3): 259 - 267. [Abstract] [Full Text] [PDF]
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M. Richards, C.-Y. Fong, S. Tan, W.-K. Chan, and A. Bongso An Efficient and Safe Xeno-Free Cryopreservation Method for the Storage of Human Embryonic Stem Cells Stem Cells, September 1, 2004; 22(5): 779 - 789. [Abstract] [Full Text] [PDF]
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K. Miyamoto, K. Hayashi, T. Suzuki, S. Ichihara, T. Yamada, Y. Kano, T. Yamabe, and Y. Ito Human Placenta Feeder Layers Support Undifferentiated Growth of Primate Embryonic Stem Cells Stem Cells, July 1, 2004; 22(4): 433 - 440. [Abstract] [Full Text] [PDF]
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J. H. Park, S. J. Kim, E. J. Oh, S. Y. Moon, S. I. Roh, C. G. Kim, and H. S. Yoon Establishment and Maintenance of Human Embryonic Stem Cells on STO, a Permanently Growing Cell Line Biol Reprod, December 1, 2003; 69(6): 2007 - 2014. [Abstract] [Full Text] [PDF]
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M. Mitalipova, J. Calhoun, S. Shin, D. Wininger, T. Schulz, S. Noggle, A. Venable, I. Lyons, A. Robins, and S. Stice Human Embryonic Stem Cell Lines Derived from Discarded Embryos Stem Cells, September 1, 2003; 21(5): 521 - 526. [Abstract] [Full Text] [PDF]
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