Bypass of Senescence, Immortalization, and Transformation of Human Hematopoietic - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

a Department of Anatomy and Cell Biology and+ U+ V: |" M1 T9 u0 z+ P* y

b Flow Cytometry Core Facility, The George Washington University Medical Center, Washington, DC, USA

Key Words. Cord blood ? CD34 ? Human telomerase catalytic subunit ? Human papillomavirus E6/E7 oncogenes ? v-H-ras ? BCR-ABL" @& _1 b' }8 O. S6 C1 ?# ~
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Correspondence: Robert G. Hawley, Ph.D., Department of Anatomy and Cell Biology, The George Washington University Medical Center, 2300 I Street NW, Washington, DC 20037, USA. Telephone: 202-994-2763; Fax: 202-994-8885; e-mail: rghawley@gwu.edu. x. _8 D( t5 q0 g9 d% Z, x. T

ABSTRACT) W8 y1 p! Q. w' j7 T

Human embryonic stem cells (hESCs) circumvent cellular senescence by expressing telomerase reverse transcriptase (hTERT) . hTERT is the catalytic subunit of telomerase, a specialized ribonucleoprotein complex that is responsible for adding telomeric DNA (repetitive TTAGGG sequences) to the ends of chromosomes to prevent shortening during replication . In this context, expression of exogenous hTERT in certain normal human somatic cell types stabilizes telomere length and allows indefinite growth . In particular, ectopic expression of hTERT has been reported to extend the lifespan of human mesenchymal stem cells and human neural progenitor cells . Candidate human hematopoietic stem cells express relatively high levels of hTERT , and telomere length analysis of human hematopoietic stem/progenitor cell sub-sets supports the hypothesis that cells with the greatest proliferative potential have the longest telomeres . Conversely, patients with aplastic anemia have short telomeres, and mutations in telomerase have been identified as the cause of hematopoietic failure .. e7 n; V, @) K8 a- y
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Previous efforts to extend the replicative capacity of human CD34  cord blood (CB) cells by retroviral-mediated expression of the hTERT gene were unsuccessful, perhaps due to transgene silencing . Therefore, to further investigate whether hTERT could be used to immortalize hematopoietic stem/progenitor sub-populations in CB samples, we used a self-inactivating (SIN) lentiviral vector backbone that we developed that directs persistent high-level expression of transgenes in hESCs and primitive human hematopoietic precursors . Besides progressive telomere shortening, it is now apparent that human cells can undergo senescence in response to various types of stress . Regardless of the senescence-initiating stimuli, the signaling pathways triggered converge to varying extents on the p53 and retinoblastoma (Rb) tumor suppressors and the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p16INK4a. Because other investigators reported that human mesenchymal stem cells could not be immortalized by hTERT alone but required combinatorial expression of the human papillomavirus type 16 (HPV16) E6 and E7 genes , which accelerate the degradation of p53 and Rb, respectively , we also attempted to prolong the lifespan of CB progenitors by transduction with an HPV16 E6/E7 lentiviral vector, separately and in conjunction with the hTERT lentiviral vector.
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MATERIALS AND METHODS

Extended Lifespan of Human CB-Derived Progenitor Cells Ectopically Expressing HPV16 E6/E7
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CD34  progenitor cells were enriched to >94% purity from mono-nuclear cell preparations of human CB samples by super paramagnetic microbead selection. The CD34  CB cells were then transduced with VSV-G glycoprotein–pseudotyped lentiviral vectors that express hTERT or HPV16 E6/E7 linked to a downstream GFP or YFP reporter gene on a bicistronic transcript. GFP  and/or YFP  CB cells were sorted to >96% purity and maintained under serum-free conditions in the presence of SCF (100 ng/ml), FL (100 ng/ml), and TPO (20 ng/ml) with or without IL-3 (20 ng/ml), conditions demonstrated to transiently support hematopoietic stem cell self-renewal divisions in vitro . Nontransduced CD34  CB cells served as controls. In all cases (n = 3), control CD34  CB cells differentiated into macrophage-like cells and underwent senescence-associated proliferation arrest after approximately 4 months in culture (Fig. 1A). Constitutive expression of hTERT failed to extend the proliferative capacity of the CD34  CB cell–derived cultures beyond this time point in repeated attempts (n = 3), and macrophage-like cells were also the predominant cell type that accumulated in these cultures (Fig. 1B), as previously reported for hTERT retroviral vector–transduced cells . On the other hand, CD34 CB-derived cells ectopically expressing HPV16 E6/E7 alone or in combination with hTERT continued to proliferate, although the cultures expressing only HPV16 E6/E7 went through a crisis period. In total, 11 CB cell lines were established, some of which have been continuously propagated in culture for more than 2 years. Cell lines obtained by the introduction of the HPV16 E6 and E7 genes were designated by the prefix "E" (two lines), and those originating from the HPV16 E6/E7-hTERT combination by "ET" (nine lines). We restricted most of our analysis to five lines:E1, E2, ET1a, ET1b, and ET2. Examination of the growth factor requirements of these five CB cell lines indicated that they all required SCF for survival and proliferation but grew optimally in the presence of SCF, FL, TPO, and IL-3. The cells were therefore routinely maintained in the four-cytokine combination.

Figure 1. Immortalization of CB progenitors by HPV16 E6/E7 with or without hTERT. (A–H): Photomicrographs of cytospin preparations after Wright-Giemsa staining (magnification x60). (A): Nontransduced CB cells at 4 months. (B): hTERT-transduced CB cells at 3 months. (C): E1 cells. (D): ET1a cells. (E): ET1b cells. (F): E2 cells. (G): ET2 cells. (H): KG1a-GFP myeloid leukemia cells. (I): Cell growth rates. The mean and SD of three experiments are shown. (J): Flow cytometric analysis of CD36 expression on ET1b and ET2 cells maintained in the absence (left panels) or presence (right panels) of erythropoietin-supplemented growth medium. The percentages of CD36  cells are indicated in the upper right quadrants. (K, L): Monocytic differentiation of CB cells. Photomicrographs of (K) ET1a and (L) E1 cells adhering to fibronectin-coated plates after nitroblue tetrazolium staining. Note that most of the ET1a cells contain formazan, the product formed by the reduction of nitroblue tetrazolium by intracellular superoxide (magnification x20). Abbreviations: APC, allophycocyanin; CB, cord blood; GFP, green fluorescent protein; HPV16, human papillomavirus type 16; hTERT, human telomerase catalytic subunit; KG1a, KG1a-GFP cells; YFP, yellow fluorescent protein.

Morphology and Cell-Surface Phenotype( Z. X" Z* e+ G+ J: }1 O
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The CB cell–derived cultures consisted of relatively homogeneous populations of nonadherent cells with round or oval nuclei located eccentrically, generally having scant cytoplasm and, in some cases (e.g., ET1a cells), microvilli-like structures on the cell surface (Figs. 1C–1G). Doubling times of the cultures supplemented with SCF, FL, TPO, and IL-3 ranged from 1.5–3 days (Fig. 1I). The surface phenotype of the CB cell lines was determined by immunofluorescence flow cytometric analysis using a panel of monoclonal antibodies directed against human hematopoietic cell-surface antigens. A summary of the analysis of the E1, E2, ET1a, ET1b, and ET2 cell lines is presented in Table 1. All of the lines expressed the leukocyte common antigen CD45, indicative of a hematopoietic origin (albeit at low levels in the case of ET1a cells). The cell lines also all expressed CD203c (basophilic granulocytes, mast cells, and their progenitors), CD71 (transferrin receptor on early erythroid cells, activated lymphocytes, monocytes, macrophages, and most dividing cells), CD44 (hyaluronan receptor on progenitors of all lineages), CD33 (myeloid progenitors, CFU-Meg, apportion of BFU-E, monocytes and mast cells, as well as activated T cells and dimly on granulocytes), and CD13 (granulocytes, monocytes, mast cells, and their progenitors). Although the CB cell lines were negative for CD34, it is notable that two of the lines (ET1a cells and ET2 cells) expressed the hematopoietic stem/progenitor cell marker CD133 . In addition, all of the cultures contained subpopulations of cells that expressed CD235a/glycophorin A (erythroid progenitor cells). In contrast, the CB cell lines expressed low or background levels of CD184 (CXCR4 "homing receptor" on CD34  progenitor cells), CD56 (natural killer cells), CD41a (gpIIb/IIIa complex on megakaryocytes), CD38 (early stages of CD34  hematopoietic stem cell lineage commitment), CD24 (B cells and granulocytes), CD19 (B cells), CD16 (natural killer cells and neutrophils), CD14 (expressed at high levels on monocytes), CD11b (M chain of the M?2 integrin expressed at varying levels on granulocytes, macrophages, myeloid-derived dendritic cells, and natural killer cells), CD3 (T-cell antigen receptor complex), CD2 (T cells and a subset of natural killer cells), and HLA-DR (antigen-presenting B cells, monocytes, and macrophages, as well as activated T cells). With the exception of the E2 cell line, which contained a subpopulation of cells that were positive, the CB cell lines were also negative for CD15 (expressed on granulocytes and to a varying degree on monocytes). Taken together, the cell-surface phenotypic and morphological properties of the CB cell lines suggested that the target cells for immortalization were multipotential progenitors of the granulocyte, monocyte-macrophage, mast cell, and erythroid lineages.

Table 1. Surface phenotype of cord blood cell lines
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Interference of the p16INK4a/Rb and p53/p21WAF1/CIP1 Pathways4 \0 E% a" ~+ q

The presence and structural integrity of the HPV16 E6/E7 and hTERT transgenes were documented by Southern blotting after digestion of genomic DNA with EcoRI plus BglII, restriction enzymes which cleave sites flanking the transgenes and the GFP/YFP reporter genes (Fig. 2A). Additional Southern blot analyses with restriction enzymes that cleave once within the vector sequences indicated two to five copies of each lentiviral vector per CB cell line (data not shown).
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Figure 2. Analysis of CB cell lines expressing HPV16 E6/E7 with or without hTERT. (A): Southern blot analysis of genomic DNA (10 μg) with the indicated probes after digestion with EcoRI plus BglII. The sizes (kb) of unrearranged transgene sequences determined by comparison with HindIII-digested  phage DNA are indicated on the right. Also shown is the 1.5-kb fragment corresponding to the endogenous BCL2 gene as restriction enzyme digestion and loading control. (B): Western blot analysis for Rb and p16INK4a. Proteins were immunoprecipitated from whole-cell lysates followed by immunoblotting. The blot was stripped and reprobed with anti–-tubulin to demonstrate that equal amounts of the respective proteins were loaded. (C): Western blot analysis for p53 and p21WAF1/CIP1 in cells treated with ( ) or without (–) actinomycin D for 24 hours. The blot was stripped and reprobed with anti–-tubulin to demonstrate that equal amounts of the respective proteins were loaded. (D): Analysis of telomerase activity by polymerase chain reaction assay. Values represent the relative ratio of the net increase of fluorescein (FL) and sulforhodamine (R) emission determined using a fluorescence plate reader. (E): Expression of hTERT stabilizes telomere length. Mean telomere length was assessed by Southern blot analysis of HinfI/RsaI-digested genomic DNA with a telomere-specific probe. The positions of size standards (kb) are indicated on the left. Abbreviations: Act. D, actinomycin D; CB, cord blood; CB1, CB9, and CB18, 1-, 9-, and 18-day cultures of primary CD34  CB cells, respectively; GFP, green fluorescent protein; high MW, high-molecular-weight control DNA; HPV16, human papillomavirus type 16; hTERT, human telomerase catalytic subunit; KG1a, KG1a-GFP cells; low MW, low-molecular-weight control DNA; Rb, retinoblastoma; TA , telomerase extract positive control; TA–, telomerase extract negative (heat-inactivated) control; YFP, yellow fluorescent protein.

We next investigated the status of the p16INK4a/Rb pathway and the p53/p21WAF1/CIP1 axis in the CB cell lines. As expected, HPV16 E7 expression in the CB cell lines resulted in reduced levels of hypophosphorylated Rb, rendering them insensitive to the increased levels of p16INK4a that accumulated (Fig. 2B). By comparison with 9-day cultures of primary CD34  CB cells, all CB cell lines showed decreased expression of the p53 target p21WAF1/CIP1, even after stimulation by actinomycin D treatment (Fig. 2C) , indicating that p53 function was compromised by the HPV16 E6 protein.
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Increased Telomerase Activity in hTERT-Expressing CB Cell Lines

CB cells stably expressing the exogenous hTERT gene exhibited high levels of telomerase activity as assayed by the telomeric repeat amplification protocol (Fig. 2D). Interestingly, the E2 cell line transduced with HPV16 E6/E7 vector alone had significant telomerase activity, and the E1 cell line exhibited telomerase activity at levels similar to early-passage CD34  CB cells. It has been reported that HPV16 E6 activates endogenous telomerase activity in precrisis human keratinocytes and mammary epithelial cells . However, analysis of a precrisis culture of HPV16 E6/E7-transduced CB cells (E5 cells at 14 weeks) revealed minimal levels of telomerase activity (Fig. 2D), suggesting that the endogenous enzyme had been reactivated in E1 and E2 cells during the immortalization process. Consistent with the notion that telomerase reactivation was associated with bypass of crisis , both E1 and E2 cell lines had substantial erosion of telomeres with mean telomere lengths of 2.9 and 2.6 kb, respectively (Fig. 2E), and widespread karyotypic abnormalities (see below). In this regard, it is noteworthy that the CB cell lines generated by cotransduction with the hTERT vector had stabilized mean telomere lengths >5.9 kb, which exceeded that of 1-day cultured primary CD34  CB cells (12.3 kb) in the case of ET1a cells (15.8 kb) transduced on the first day of culture (Fig. 2E).  b* C  J, Y/ N( c1 S0 J. t
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Clonal Outgrowth of HPV16 E6/E7 Plus hTERT Immortalized CB Cells Without Widespread Genomic Instability5 K9 q3 [" x) a& A" z

After 28–76 weeks of culture, the CB cell lines were analyzed by SKY, chromosome G-banding, and FISH with centromere-specific probes. Although the CB cell populations were not deliberately cloned, these analyses indicated that most of them were clonal (Fig. 3, Table 2). The exceptions were the E1 and E2 cell lines obtained by transduction with HPV16 E6/E7 alone and the ET1b cell line, in which the hTERT gene was introduced at week 44 of culture. All three of these cell lines were highly abnormal and carried additional distinctive karyotypic changes indicating that they were oligoclonal. By comparison, the CB cell lines immortalized by successive coexpression of HPV16 E6/E7 and hTERT within 4 weeks of culture were near diploid and exhibited only one or two structural or numerical chromosomal changes.1 c  A; H  h2 u" I; H
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Figure 3. Spectral karyotyping (left panels) and G-banded (right panels) chromosome analysis of cord blood cell lines immortalized by HPV16 E6/E7 and hTERT showing limited aneuploidy and rearrangements. (A): ET1a. (B): ET2. (C): ET3. (D): ET4. (E): ET5. Structural and numerical chromosomal abnormalities are indicated by arrows. See Table 2 for a detailed description of the karyotypes. Abbreviations: HPV16, human papillomavirus type 16; hTERT, human telomerase catalytic subunit.
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Table 2. Karyotype of cord blood cell lines9 n  M8 K( J7 N+ ]# J! [3 _

Differentiation Potential of Immortalized CB Cells

Based on their phenotype and growth factor requirements, the immortalized CB cells most closely resembled myeloerythroid/mast cell progenitors . Although mast cell progenitors have generally been considered to be a separate lineage, CD203c is also expressed on basophilic granulocytes and their progenitors, raising the possibility that the CB cell lines originated from more primitive common committed CD34  progenitors . Indeed, it was previously shown that TPO played an important role in concert with SCF in the development of mast cells from CD34  multilineage colony-forming cells that also had the potential to differentiate into neutrophil/macrophage/mast cell/erythroid lineages, neutrophil/macrophage/mast cell lineages, or neutrophil/mast cell lineages . TPO is also known to enhance erythroid progenitor production from CD34  bone marrow and CB cells .7 D$ y) V! `( Z6 q% d+ f
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Whether the CB cell lines can be efficiently induced to terminally differentiate into functional mast cells, erythrocytes, granulocytes, and/or monocyte-macrophages by treatment with various physiologic or chemical/pharmacologic agents will require further investigation. However, pilot experiments suggest that the CB cell lines have a certain degree of erythroid and myeloid differentiative potential. Some shifting to an erythroblastic phenotype was observed when the CB cell lines were cultured under erythropoiesis-supportive conditions , as evidenced by slightly increased expression of the CD36 antigen (Fig. 1J) . Moreover, when the immortalized CB cells were subjected to a myeloid differentiation regimen , in the best example (ET1a cells) up to 90% of the cells acquired the ability to adhere to fibronectin, approximately 30% of which were capable of superoxide-dependent nitroblue tetrazolium reduction reflective of terminal monocytic differentiation (Figs. 1K, 1L).8 A" b6 e3 ^2 h8 Z* W( l3 b

In Vivo Growth Potential of E6/E7 Plus hTERT-Expressing CB Cells

The ET1a and ET2 CB cell lines were transduced with retroviral vectors coexpressing the v-H-ras  or BCR-ABL  oncogenes and the bacterial neomycin phosphotransferase (neo) gene or with a control vector expressing the neo gene alone. Geneticin-resistant bulk populations of ET1a/Neo and ET2/Neo cells did not engraft or grow in sublethally irradiated (250-cGy) immunodeficient NOD/SCID mice after i.v. or s.c. injection, respectively, during observation periods of 28 weeks (Table 3). Disseminated (bone marrow, spleen, liver, and peripheral blood) or solid tumor growth was observed, however, within 14 weeks after i.v. or s.c. injection of v-H-ras–transduced or BCR-ABL–transduced E6/E7 plus hTERT-expressing cell populations. By comparison, malignant growth of i.v.- or s.c.-injected GFP-expressing KG1a myeloid leukemia cells occurred within 8 weeks.
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Table 3. Evaluation of tumor formation and engraftment of CB cell lines in immunodeficient NOD/SCID mice

DISCUSSION
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We thank Joseph Molete for technical assistance. This work was supported in part by National Institutes of Health grants R01HL65519, R01HL66305, and R24RR16209.
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DISCLOSURES/ a; m" @- @( D( t7 i1 G' v
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R.G.H. receives royalties derived from the licensing of gene transfer technology (MSCV Retroviral Expression System) for research purposes to Clontech.
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