Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, S.-J.
Right arrow Articles by Honig, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, S.-J.
Right arrow Articles by Honig, G. R.

Stem Cells 2002;20:428-437 www.StemCells.com
© 2002 AlphaMed Press

Hematopoietic Progenitor Cells Derived from Embryonic Stem Cells: Analysis of Gene Expression

Shi-Jiang Lu, Chengshi Quan, Fei Li, Loyda Vida, George R. Honig

Department of Pediatrics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA

Key Words. ES cells • HSC • Cell cycle • CD34+ cells • Gene expression

Shi-Jiang Lu, Ph.D., Department of Pediatrics, M/C 856, University of Illinois at Chicago, 840 South Wood Street, Chicago, Illinois 60612, USA. Telephone: 312-413-5766; Fax: 312-413-1526; e-mail sjlu{at}uic.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rhesus monkey embryonic stem (ES) cells undergo differentiation in vitro to generate hematopoietic progenitor cells. Our previous studies demonstrated a high degree of similarity in the expression of genes associated with hematopoietic differentiation, homing, and engraftment in CD34+ and CD34+CD38- cells from rhesus monkey ES cells and from fresh or cultured bone marrow (BM). In the present study, we compared the expression patterns of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDIs) in these cells. The expression of genes for cyclins, CDKs, and CDIs was similar among the hematopoietic progenitor cells of different origins, with only minor differences. Differentially expressed genes were also analyzed in CD34+ lineage-negative cells derived from mouse ES cells and from BM. No difference or totally divergent results were obtained with the latter system, suggesting that this variation may be species specific. We observed, however, that CD34+ and CD34+CD38- cells derived from ES cells expressed embryonic {varepsilon} and {zeta} as well as {alpha}, ß, and {gamma} globin genes, whereas no expression of embryonic globins could be detected in the cell preparations from BM. Moreover, erythroblast-enriched CD34- cells derived from 4- or 5-week ES cell differentiation cultures also expressed embryonic, fetal, and adult globin genes, with greater ß gene expression, but otherwise were identical to those of the more primitive CD34+ cells derived from 2-week ES cultures. These latter observations may reflect the presence of heterogeneous cell populations within the cell fractions that were compared, or they may represent variability among ES-cell-derived hematopoietic stem cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Long-term cultures of pluripotent embryonic stem (ES) cells have been established from a variety of mammalian species, including nonhuman primates and man [1–4]. Under appropriate culture conditions, ES cells undergo differentiation in vitro to form hematopoietic precursors [5–8]. Flow cytometry analyses of rhesus monkey ES-cell-derived hematopoietic progenitor cells demonstrated that a substantial percentage of these cells expressed CD34 antigen and had morphological features of undifferentiated blast cells [7]. The hematopoietic character of these precursors is further supported by the demonstration that they express genes associated with early hematopoietic differentiation, and that they exhibit morphological findings of erythroid and myeloid lineages among their progeny cells [7]. These results parallel those from observations of murine and human ES cell differentiation in vitro [5,6,8].

Among the distinctive characteristics of hematopoietic stem cells (HSCs) is their ability to repopulate bone marrow (BM)-ablated animals. Murine HSCs derived from ES cell differentiation in vitro, however, lack long-term reconstitution potential when transplanted into adult recipient mice [9,10]. The failure of ES-cell-derived HSCs to engraft could be related to a failure of these precursors to express appropriate molecules that are critical for engraftment. To explore this possibility, we compared the expression patterns of multiple genes associated with hematopoietic differentiation, HSC homing, and engraftment in CD34+ and CD34+CD38- cells derived from rhesus monkey ES cells with those isolated from fresh or cultured adult BM. These studies demonstrated a remarkable degree of similarity in the expression patterns of these genes in both the CD34+ and CD34+CD38- cells, with only few exceptions [11]. Most notably, the message of the flt3 gene was undetectable in rhesus monkey ES-cell-derived CD34+ and CD34+CD38- cells, whereas substantial flt3 expression was observed in the corresponding cells from fresh BM and in CD34+ cells from cultured BM. The expression of both integrin {alpha}L and interleukin-6 (IL-6) receptor genes also was very low, or negligible, in CD34+CD38- cells derived from rhesus monkey ES cell cultures, whereas corresponding cell preparations isolated from fresh BM of rhesus monkeys expressed high levels of these genes. In parallel with these observations, the flt3, integrin {alpha}L, and IL-6 receptor genes displayed robust expression in CD34+ lineage-negative (Lin-) cells from murine BM, but had no apparent expression in CD34+Lin- cells from murine ES cells [11].

Several recent studies have demonstrated that the entry of cultured HSCs into the active cell cycle dramatically reduces their homing ability, and subsequently, their long-term repopulating potential [12–18]. Eukaryotic cell cycle progression is regulated by the cyclin-dependent kinases (CDKs), whose activities are controlled by multiple mechanisms, including cyclin binding, CDK phosphorylation and dephosphorylation, and binding of CDK inhibitors (CDIs) [19]. CDI p21 protein has been shown to maintain hematopoietic stem cell quiescence by controlling their entry into the cell cycle [20]. The tumor suppressor gene, WT1, induces the expression of p21 and also promotes the quiescence of CD34+CD38- cells [21]. CDI p27 does not affect hematopoietic stem cell cycling and self-renewal, but it markedly alters progenitor proliferation and pool size in the knockout mouse model [22]. These findings demonstrate that cell cycle control genes also play an important role in regulating hematopoietic stem cell self-renewal and differentiation.

In the present study, we examined the expression patterns of cyclins, CDKs, and CDIs, HoxB4, and Bcrp1/ABCG2 in hematopoietic progenitor cells derived from rhesus monkey ES cell cultures and from adult rhesus BM, using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). We also examined the expression patterns of embryonic, fetal, and adult globin genes. Most HSCs in BM are believed to be in the G0 resting state, whereas ES-cell-derived hematopoietic precursors, which have been exposed to hematopoietic growth factors included in the culture media, are in active growth. These differences could confound comparisons of gene expression between them. To circumvent this potential limitation, we also examined gene expression of CD34+ and CD34+CD38- cells purified from BM that had been maintained under the same culture conditions that were employed for ES cell hematopoietic differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of Rhesus Monkey CD34+ and CD34+CD38- Cells
The maintenance and induction of hematopoietic differentiation of R366.4 rhesus monkey ES cells and purification of CD34+ and CD34+CD38- cells were as previously described [7,11]. In brief, CD34+ cells were isolated from differentiated ES colonies with biotinylated anti-human CD34+ antibody (clone 12.8; Baxter Healthcare Corporation; Deerfield, IL; http://www.baxter.com) using a Coulter flow cytometer. For CD34+CD38- cell preparation, CD38+ cells were first depleted using anti-human CD38 antibody (clone OKT10; American Type Culture Collection [ATCC]; Manassas, VA; http://www.atcc.org), then CD34+CD38- cells were sorted by two color parameters with a Coulter flow cytometer.

BM from rhesus monkeys (aged 2-8 years) was obtained through the animal tissue distribution network from the Wisconsin Regional Primate Center and from the Biologic Resources Laboratory of the University of Illinois at Chicago. Each BM sample was aliquoted into two parts. One part was directly used for the isolation of CD34+ and CD34+CD38- cells (fresh); the other aliquot was incubated in ES differentiation medium for 3 days, then sorted for the purification of CD34+ and CD34+CD38- cells (cultured). Both CD34+ and CD34+CD38- cells, with a purity of >95%, were obtained from rhesus monkey ES cell cultures and fresh and cultured BM. The fluorescence-activated cell-sorting profiles that define the gates for the purification of the CD34+CD38- cells have been reported previously [11].

Enrichment of Rhesus Monkey Erythroblasts from ES Cell Cultures
Rhesus monkey ES cells were grown under differentiation conditions as described [7], then were replated and cultured for erythrocyte formation in vitro. After 4 or 5 weeks of culture, erythroblast clusters were collected by rinsing them off the feeder cells with fresh medium. CD34+ cells were depleted by anti-human CD34+ antibody with Miltenyi magnetic beads (Miltenyi Biotech; Auburn, CA; http://miltenyibiotec.com). Fifteen percent to 20% of the CD34- fractions were found to be erythroblasts by microscopic examination of stained preparations.

Purification of Mouse CD34+Lin- Cells
The mouse D3 ES line was obtained from the ATCC. The maintenance and induction of hematopoietic differentiation of the D3 ES cells and CD34+Lin- cell purification were as described previously [11]. Briefly, at day 13 of differentiation, hematopoietic-like clusters were collected and stained with fluorescein isothiocyanate-conjugated anti-mouse CD34+ (clone RAM34; PharMingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen) and biotinylated anti-mouse lineage panel (containing anti-CD3e, CD11b, CD45R/B220, Ly6G, and TER-119; Baxter) coupled with streptavidin-phycoerythrin. CD34+Lin- cells were sorted with a Coulter flow cytometer. BM cells were obtained by flushing the tibias and femurs of strain 129/sv mice (The Jackson Laboratory; Bar Harbor, ME; http://www.jax.org). CD34+Lin- cells were purified from mononuclear cells as described above. CD34+Lin- cells with a purity of >95% were obtained from both D3 ES cell cultures and mouse BM.

Cytoplasmic RNA Isolation and cDNA Pool Construction
Cytoplasmic RNA isolation and cDNA pool construction for rhesus monkey CD34+ cells, CD34+CD38- cells, and CD34- erythroblasts and mouse CD34+Lin- cells were as described previously [11]. cDNA pools generated by the SMART (Clontech; Palo Alto, CA) procedure have been shown to preserve the relative abundance relationship in original mRNA populations [23], and this procedure has successfully been used in the construction of cDNA pools using less than 1,000 cells [24].

The DNA templates in cDNA pools of CD34+ and CD34+CD38- cells derived from rhesus monkey ES cells and fresh and cultured BM were adjusted to equal amounts based on the relative expression levels of the hypoxanthine phosphoribosyltransferase (HPRT) gene, as described [11]. Similarly, the DNA template quantities in the cDNA pools of CD34+Lin- cells derived from murine ES cells and BM were equalized based on the signal intensities of the {alpha}-tubulin gene.

Gene Expression Quantification by Semiquantitative PCR
Inasmuch as many of the genes we undertook to study have not been cloned from the rhesus monkey, whenever rhesus monkey-specific sequences were unavailable, we employed the sequences of the corresponding human genes to design our PCR primers, assuming that the generally close homology between human and rhesus monkey would compensate for minor differences in gene sequences in PCR amplification [25]; this approach has been applied successfully for a large number of other genes in our previous study and other studies [7,11,25,26]. The sense and antisense primer sequences and the corresponding cDNA PCR product sizes are shown in Table 1Go. Cytoplasmic RNA (5 µg) from fresh rhesus and murine BM and from K562 cells was reverse transcribed to single-stranded cDNA and used to optimize PCR conditions for the genes we analyzed. The conditions for PCR amplification were as described [11] with annealing temperatures and concentrations of MgCl2 for each specific gene shown in Table 1Go. Ten µl of PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. The relative expression levels in cDNA from ES cells and cultured BM cells were estimated visually, according to the relative band intensities compared with those of cells directly isolated from fresh BM. The expression of genes in mouse CD34+Lin- cells was analyzed similarly.


View this table:
[in this window]
[in a new window]
  		Table 1. PCR primers and conditions for gene analysis
 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CDKs
We examined the expression of a number of CDK genes that have been shown to be positive regulators of the cell cycle [19]; the results are summarized in Table 2Go and Figure 1Go. All CDKs analyzed were shown to be expressed at high levels in both CD34+ and CD34+CD38- cells of all origins, although greater or lower expression of some CDKs was observed. A moderately lower CDK4 expression was observed in CD34+ and CD34+CD38- cells derived from cultured BM, whereas a greater level of CDK4 was seen in ES-cell-derived CD34+, but not CD34+CD38- cells, compared with cells from fresh BM. CD34+ cells from ES cell differentiation and cultured BM showed a lower level of CDK5 gene expression, but no differences were found in any of the CD34+CD38- cell preparations. No significant difference in expression of CDK1 (cdc2), CDK2, and CDK6 was observed among progenitor cells isolated from ES cells and from fresh and cultured BM.


View this table:
[in this window]
[in a new window]
  		Table 2. Expression of genes in CD34+ and CD34+CD38- cells derived from rhesus monkey ES cells and BM with and without added cytokines
 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Representative analyses of gene expression in CD34+ and CD34+CD38- cells derived from rhesus monkey ES cell differentiation (ES), fresh BM (BM), and BM cultured with cytokines for 3 days (BM + C). Cytoplasmic RNA from CD34+ and CD34+CD38- cells was used to construct cDNA pools, and the expression of genes was examined by semiquantitative PCR. The number at the top of each lane indicates the amount (µl) of cDNA used in the 50-µl PCR reaction. M = 1 kb DNA marker; WBM = unfractionated rhesus monkey adult BM.

 
Cyclins
Cyclins are members of a family of structurally related proteins that bind to and activate CDKs. The formation of different CDK-cyclin complexes is required to phosphorylate several protein substrates that are important to drive cell cycle events [19]. Our analysis of cyclin expression in hematopoietic progenitor cells derived from ES cells and BM demonstrated similar expression patterns, with a few exceptions: cyclin A was expressed at relatively high levels in CD34+ cells of all origins, whereas the message of cyclin A was only detected in CD34+CD38- cells derived from ES cells; no detectable mRNA was found in the comparable preparation from BM. A low level of cyclin B expression was detected in CD34+ cells of all three sources, and substantial levels of cyclin B message were expressed in CD34+CD38- cells derived from ES cells and from cultured BM, but a somewhat lower level was found in cells derived from fresh BM.

The expression of cyclin D family members showed quite different patterns. Very low levels of cyclin D1 message were found in both CD34+ and CD34+CD38- progenitors from all origins, whereas a moderate level of cyclin D2 expression was expressed in these cell preparations and a relatively greater degree of expression was observed in CD34+CD38- cells from ES cells. Low levels, but with no difference in cyclin D3 expression, were detected in both CD34+ and CD34+CD38- cells from all sources except CD34+ cells from cultured BM, which showed a lower expression than the corresponding cell preparation from fresh BM. High levels of cyclin E message were expressed in hematopoietic progenitors from ES and BM, and a somewhat higher expression was observed in CD34+CD38- cells of ES cell origin. Similarly, very high levels of cyclin G1 expression were found in both CD34+ and CD34+CD38- cells; the CD34+CD38- cells from ES differentiation were found to express a higher level of cyclin G1 than those from BM.

CDIs
CDIs can be divided into two families, the INK4 family and the CIP/KIP family. Genes of the INK4 family encode inhibitors of CDK4, and four of these (p15, p16, p18, and p19) have been identified. The CIP/KIP family includes p21, p27, and p57 [27]. The expression of CDIs in hematopoietic progenitor cells from ES and BM showed quite different expression patterns. There were very low to negligible levels of p15 gene message in CD34+ and CD34+CD38- cells of all origins, although a clear positive signal was obtained from the rhesus monkey whole BM control. The message of another INK4 family gene, p16, was only observed in CD34+ cells derived from ES cells, and no detectable level of p16 expression was found in CD34+CD38- cells from all sources and in CD34+ cells from BM. A very low level of p16 mRNA was detected in unfractionated BM. No significant level of p18 message was expressed in hematopoietic progenitor cells derived from ES cells, whereas a substantial level of p18 was detected in both CD34+ and CD34+CD38- cells from fresh BM; a lower, but clearly detectable, level of p18 message was found in corresponding cell preparations from cultured BM. In contrast to other members of the INK4 gene family, the expression of the p19 gene was found to be at very high levels in both CD34+ and CD34+CD38- cells of all origins.

Substantial levels of p21 message were expressed in CD34+ and CD34+CD38- cells from ES cells and fresh BM, and a lower expression of p21 was observed in the corresponding cell preparation from cultured BM. Similar patterns of p27 expression were demonstrated for both CD34+ and CD34+CD38- cells, and cells from ES and cultured BM showed lower p27 expression than those from fresh BM. The expression of p57, which is closely related to p27, was quite different between CD34+ and CD34+CD38- cells. Similar to p27, lower p57 expression was found in CD34+ cells derived from ES cells and from cultured BM. In contrast, the p57 message was only detectable in ES-cell-derived CD34+CD38- cells, and no p57 mRNA was expressed in the cell preparations from BM. The expression of WT1, a tumor suppressor gene that influences cell cycle events by regulating p21 expression [21], was also examined. Lower expression was detected in CD34+ cells derived from ES and cultured BM than in those from fresh BM. Strikingly, no detectable message of WT1 was expressed in CD34+CD38- cells from ES or cultured BM, whereas a substantial level of WT1 expression was demonstrated in the same cell preparation from fresh BM.

Globin Genes
In our previous studies, we demonstrated the expression of {alpha}-, ß-, and {gamma}-globin genes in hematopoietic progenitor cells from all origins [11]. In this study, we also examined the messages of two embryonic globin genes, {zeta} and {varepsilon}, in both CD34+ and CD34+CD38- cells. As shown in Table 2Go, very high levels of {zeta}- and {varepsilon}-globin gene expression were detected in both CD34+ and CD34+CD38- cells derived from ES cells, and as expected, no detectable message was found in the cell preparations from BM. We also analyzed the expression patterns of globin genes in erythroblast-enriched CD34- cells derived from 4- and 5-week ES cell differentiation cultures. As shown in Figure 2Go, a greater ß-globin gene expression was found in erythroblast-enriched CD34- cells derived from 4- and 5-week cultures than in CD34+ cells derived from 2-week cultures. However, similar expression levels of the other four globin genes were observed in erythroblast-enriched CD34- cells and CD34+ cells.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 2. Analyses of globin gene expression in CD34+ cells derived from 14-day and erythroblast-enriched CD34- cells derived from 28- and 35-day rhesus monkey ES cell differentiation cultures. Cytoplasmic RNA from CD34+ and CD34- cells was used to construct cDNA pools, and the expression of adult, fetal, and embryonic globin genes was examined by semiquantitative PCR. HPRT was used to equalize the amounts of cDNA. The number at the top of each lane indicates the amounts (ml) of cDNA used in the 50-ml PCR reaction. M = 1 kb plus DNA marker (BRL); P = positive controls: RNA from rhesus monkey adult BM for the {alpha}-, ß-, and {gamma}-globin genes; RNA from leukemia K562 cells for the {zeta}- and {varepsilon}- globin genes.

 
HoxB4 and Bcrp1 Genes
Previous studies have suggested that HoxB4 plays an important role in HSC proliferation and differentiation. Enforced expression of HoxB4 has been reported to lead to the expansion of adult HSCs ex vivo and the production of HSCs with amplified competitive repopulating ability in vivo with no apparent associated leukemic potential [28–31]. We, therefore, also examined HoxB4 gene expression in hematopoietic progenitor cells derived from ES cells and BM. We observed no apparent difference in HoxB4 expression; a low, but detectable, level of HoxB4 message was present in the CD34+ cells of all origins (Table 2Go). The expression of HoxB4 was very low or negligible in CD34+CD38- cells, especially in the cells from ES differentiation. We also examined the expression of Bcrp1/ABCG2, which has been shown to be a useful marker for the "side-population" cells of BM [32,33]. A high level of message could be detected in both CD34+ and CD34+CD38- cells of all three sources, and no significant differences in expression levels were observed among CD34+ cells of all origins; a somewhat higher level of expression was seen in CD34+CD38- cells derived from fresh BM (Table 2Go).

Gene Expression in Mouse CD34+Lin- Cells
Parallel studies to those of rhesus monkey HSC gene expression were performed to compare their expression in murine hematopoietic precursors. For these studies, we purified hematopoietic CD34+Lin- cells from strain 129/sv mouse BM and from differentiation cultures of mouse D3 ES cells. These experiments showed no apparent correlation of expression for p18, p27, WT1, and cyclin A in hematopoietic progenitor cells derived from the mouse and from the monkey (Fig. 3Go and Table 3Go). A somewhat lower expression of cyclin A gene was observed in CD34+Lin- cells from murine ES cells compared with that in corresponding cells from murine BM. We also did not observe a lower WT1 gene expression in CD34+Lin- cells derived from murine ES cell cultures, and with the mouse system, there was, rather, a greater level of expression of this gene than with CD34+Lin- cells purified from BM. Expression levels of the p18 and p27 genes were also examined, and no difference could be detected for the p27 gene between CD34+Lin- cells from murine ES cells and those harvested from BM; a somewhat greater level of p18 expression was observed in the corresponding cells from murine BM.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. Representative analyses of gene expression in CD34+Lin- cells derived from mouse D3 ES cell differentiation (ES) and BM. Cytoplasmic RNA from CD34+Lin- cells was used to construct cDNA pools, and the expression of genes was examined by semiquantitative PCR. The number at the top of each lane indicates the amount (µl) of cDNA pool used in the 50-µl PCR reaction. M = 1 kb DNA marker (BRL); WBM = unfractionated strain 129/sv mouse BM.

 

View this table:
[in this window]
[in a new window]
  		Table 3. Gene expression in CD34+Lin- cells derived from mouse ES cells and BM
 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings demonstrate a high and generally consistent level of expression of cyclins and CDKs in CD34+ and CD34+CD38- cells harvested from BM and in those derived from ES cell differentiation. The analysis of CDI genes showed heterogeneous patterns of expression, and some differences were observed for several of these genes. Parallel studies of the expression analysis of these genes in CD34+Lin- cells derived from mouse ES cell differentiation and from adult BM, however, demonstrated no apparent difference or totally divergent results, suggesting that the observed differences are likely to be species specific, and presumably, do not reflect changes that determine the biological functions of HSCs derived from ES cell cultures.

Our previous [11] and present findings demonstrate, overall, a remarkable degree of similarity in the expression patterns of genes that are associated with hematopoietic differentiation, HSC homing and engraftment, and cell cycle regulation in both CD34+ and CD34+CD38- cells from ES cells and BM. These findings speak against the notion that altered expression of these genes has a determinative role in the failure of ES-derived HSCs to engraft in the BM of adult recipients.

As an alternative explanation, it has been proposed that ES-cell-derived HSCs may retain the properties of ES cells, which in turn, may require specific elements of the fetal environment for engraftment to take place. Supporting this idea are observations showing that when mouse yolk sac progenitor cells were transplanted into livers of newborn pups, the presence of long-term repopulating stem cells could be demonstrated, whereas the same cells transplanted into adult animals exhibited no such repopulating potential [34]. Also consistent with the latter hypothesis are our observations that both CD34+ and CD34+CD38- cells derived from fresh and cultured BM only expressed adult and fetal globin genes, whereas the same cell preparations from ES cell differentiation expressed some adult, but mainly fetal and embryonic, globin genes. A somewhat greater ß-globin gene expression could be detected in erythroblast-enriched CD34- cells derived from 4- and 5-week cultures, compared with CD34+ cells derived from 2-week cultures; however, there were remarkably similar expressions of the embryonic and fetal globin genes in erythroblast-enriched CD34- cells and CD34+ cells, resembling the expression patterns of cells derived from the mouse yolk sac [35]. These results suggest that both embryonic hematopoietic progenitor cells and hematopoietic cells derived from rhesus monkey ES cell differentiation exhibit similar gene expression patterns. Cells derived from ES cell differentiation are likely to contain subpopulations of hematopoietic precursors, possibly at varying stages of differentiation, and to understand the full significance of these observed results, it will be important to determine if ß-chain gene expression in cells from ES cell differentiation is limited to a discrete subpopulation of hematopoietic precursors. However, in either case, the preponderance of evidence suggests that ES cell differentiation in vitro has been largely blocked at the embryonic stage, with few definitive HSCs having been generated.

HoxB4, a member of the homeobox gene family, has been shown to be abundantly expressed in primitive hematopoietic cells, but then declines with lineage-specific terminal differentiation [36–38]. We found very low, but detectable, levels of HoxB4 message in CD34+ and CD34+CD38- cells derived from differentiated ES cells. It has been reported that overexpression of the HoxB4 gene induced rapid, extensive expansion of HSCs derived from mouse BM ex vivo [31] and produced stem cells that had amplified competitive repopulating ability in transplantation experiments [28–30]. Overexpression of HoxB4 also resulted in a significantly greater number of progenitors of mixed erythroid/myeloid colonies and of definitive, but not primitive, erythroid colonies derived from ES cell differentiation [35, 39]. Mouse embryoid bodies derived from HoxB4-transduced ES cells showed a significantly greater adult ß-globin expression [35,39]. When transplanted into lethally irradiated adult mice, these cells homed to and engrafted in the BM, with long-term and multilineage contribution to the peripheral blood [35]. These findings demonstrate that enforced expression of the HoxB4 gene enhances hematopoietic differentiation of mouse ES cells in vitro, and they suggest that primitive HSCs from ES cells are poised to become definitive HSCs in response to the appropriate signals. These observations may provide a fruitful direction for further study of primate ES cell differentiation in vitro.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Dr. James A. Thomson (Wisconsin Regional Primate Research Center, University of Wisconsin) for providing the rhesus monkey ES cells for this study; the Wisconsin Regional Primate Center and the Biologic Resources Laboratory of the University of Illinois at Chicago for providing rhesus monkey bone marrow; Dr. Jianxun Li and Ximing Zhou (College of Dentistry, University of Illinois at Chicago) for assistance with graphic photo imaging; and Dr. Karen Hagen (Research Resources Center, University of Illinois at Chicago) for flow cytometry analyses.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156.[CrossRef][Medline]

  2. Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA 1995;92:7844–7848.[Abstract/Free Full Text]

  3. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.[Abstract/Free Full Text]

  4. Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.[CrossRef][Medline]

  5. Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995;7:862–869.[CrossRef][Medline]

  6. Weiss MJ, Orkin SH. In vitro differentiation of murine embryonic stem cells. New approaches to old problems. J Clin Invest 1996;97:591–595.[Medline]

  7. Li F, Lu S-J, Vida L et al. Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro. Blood 2001;98:335–342.[Abstract/Free Full Text]

  8. Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2001;98:10716–10721.[Abstract/Free Full Text]

  9. Hole N, Graham GJ, Menzel U et al. A limited temporal window for the derivation of multilineage repopulating hematopoietic progenitors during embryonal stem cell differentiation in vitro. Blood 1996;88:1266–1276.[Abstract/Free Full Text]

  10. Palacios R, Golunski E, Samaridis J. In vitro generation of hematopoietic stem cells from an embryonic stem cell line. Proc Natl Acad Sci USA 1995;92:7530–7534.[Abstract/Free Full Text]

  11. Lu S-J, Li F, Vida L et al. Comparative gene expression in hematopoietic progenitor cells derived from embryonic stem cells. Exp Hematol 2002;30:58–66.[CrossRef][Medline]

  12. Glimm H, Oh IH, Eaves CJ. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0). Blood 2000;96:4185–4193.[Abstract/Free Full Text]

  13. Szilvassy SJ, Meyerrose TE, Grimes B. Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells. Blood 2000;95:2829–2837.[Abstract/Free Full Text]

  14. van der Loo JC, Ploemacher RE. Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors. Blood 1995;85:2598–2606.[Abstract/Free Full Text]

  15. Thornley I, Nayar R, Freedman MH et al. Differences in cell cycle kinetics of candidate engrafting cells in human bone marrow and mobilized peripheral blood. Exp Hematol 2001;29:525–533.[CrossRef][Medline]

  16. Gothot A, van der Loo JC, Clapp DW et al. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34(+) cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641–2649.[Abstract/Free Full Text]

  17. Summers YJ, Heyworth CM, de Wynter EA et al. Cord blood G(0) CD34+ cells have a thousand-fold higher capacity for generating progenitors in vitro than G(1) CD34+ cells. STEM CELLS 2001;19:505–513.[Abstract/Free Full Text]

  18. Jetmore A, Plett PA, Tong X et al. Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients. Blood 2002;99:1585–1593.[Abstract/Free Full Text]

  19. Morgan DO. Principles of CDK regulation. Nature 1995;374:131–134.[CrossRef][Medline]

  20. Cheng T, Rodrigues N, Shen H et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 2000;287:1804–1808.[Abstract/Free Full Text]

  21. Ellisen LW, Carlesso N, Cheng T, Scadden DT et al. The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells. EMBO J 2001;20:1897–1909.[CrossRef][Medline]

  22. Cheng T, Rodrigues N, Dombkowski D et al. Stem cell repopulation efficiency but not pool size is governed by p27(kip1). Nat Med 2000;6:1235–1240.[CrossRef][Medline]

  23. Zhumabayeva B, Diatchenko L, Chenchik A et al. Use of SMART-generated cDNA for gene expression studies in multiple human tumors. Biotechniques 2001;30:158–163.[Medline]

  24. Zheng J, Shen W, He DZ et al. Prestin is the motor protein of cochlear outer hair cells. Nature 2000;405:149–155.[CrossRef][Medline]

  25. Gomes I, Sharma TT, Mahmud N et al. Highly abundant genes in the transcriptosome of human and baboon CD34 antigen-positive bone marrow cells. Blood 2001;98:93–99.[Abstract/Free Full Text]

  26. Kayo T, Allison DB, Weindruch R et al. Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. Proc Natl Acad Sci USA 2001;98:5093–5098.[Abstract/Free Full Text]

  27. Sherr CJ. The INK4a/ARF network in tumour suppression. Nat Rev Mol Cell Biol 2001;2:731–737.[CrossRef][Medline]

  28. Sauvageau G, Thorsteinsdottir U, Eaves CJ et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 1995;9:1753–1765.[Abstract/Free Full Text]

  29. Antonchuk J, Sauvageau G, Humphries RK. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp Hematol 2001;29:1125–1134.[CrossRef][Medline]

  30. Thorsteinsdottir U, Sauvageau G, Humphries RK. Enhanced in vivo regenerative potential of HOXB4-transduced hematopoietic stem cells with regulation of their pool size. Blood 1999;94:2605–2612.[Abstract/Free Full Text]

  31. Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 2002;109:39–45.[CrossRef][Medline]

  32. Zhou S, Schuetz JD, Bunting KD et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.[CrossRef][Medline]

  33. Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. Blood 2002;99:507–512.[Abstract/Free Full Text]

  34. Yoder MC, Hiatt K, Dutt P et al. Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity 1997;7:335–344.[CrossRef][Medline]

  35. Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 2002;109:29–37.[CrossRef][Medline]

  36. Sauvageau G, Lansdorp PM, Eaves CJ et al. Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci USA 1994;91:12223–12227.[Abstract/Free Full Text]

  37. Magli MC, Largman C, Lawrence HJ. Effects of HOX homeobox genes in blood cell differentiation. J Cell Physiol 1997;173:168–177.[CrossRef][Medline]

  38. Pineault N, Helgason CD, Lawrence HJ et al. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol 2002;30:49–57.[CrossRef][Medline]

  39. Helgason CD, Sauvageau G, Lawrence HJ et al. Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro. Blood 1996;87:2740–2749.[Abstract/Free Full Text]

Received April 22, 2002; accepted for publication July 2, 2002.



This article has been cited by other articles:


Home page
 Stem CellsHome page
D. Rajesh, N. Chinnasamy, S. M. Mitalipov, D. P. Wolf, I. Slukvin, J. A. Thomson, and A. F. Shaaban
Differential Requirements for Hematopoietic Commitment Between Human and Rhesus Embryonic Stem Cells
Stem Cells, February 1, 2007; 25(2): 490 - 499.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
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]

Home page
 BloodHome page
S.-J. Lu, F. Li, L. Vida, and G. R. Honig
CD34+CD38- hematopoietic precursors derived from human embryonic stem cells exhibit an embryonic gene expression pattern
Blood, June 1, 2004; 103(11): 4134 - 4141.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
K. Umeda, T. Heike, M. Yoshimoto, M. Shiota, H. Suemori, H. Y. Luo, D. H. K. Chui, R. Torii, M. Shibuya, N. Nakatsuji, et al.
Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro
Development, April 15, 2004; 131(8): 1869 - 1879.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. Cerdan, A. Rouleau, and M. Bhatia
VEGF-A165 augments erythropoietic development from human embryonic stem cells
Blood, April 1, 2004; 103(7): 2504 - 2512.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, S.-J.
Right arrow Articles by Honig, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, S.-J.
Right arrow Articles by Honig, G. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.peprotech.com/