HOXB4 Overexpression Promotes Hematopoietic Development by Human Embryonic Stem - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:06 |只看该作者 |倒序浏览 |打印

作者：Kristian M. Bowles, Ludovic Vallier, Joseph R. Smith, Morgan R. J. Alexander, Roger A. Pedersen作者单位：Department of Surgery, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom

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      【摘要】8 u. k; H7 J3 h! }1 h
   Human embryonic stem cells (hESCs) are a potential source of hematopoietic cells for therapeutic transplantation and can provide a model for human hematopoiesis. Culture of hESCs on murine stromal layers or in stromal-free conditions as embryoid bodies results in low levels of hematopoietic cells. Here we demonstrate that overexpression of the transcription factor HOXB4 considerably augments hematopoietic development of hESCs. Stable HOXB4-expressing hESC clones were generated by lipofection and could be maintained in the undifferentiated state for prolonged passages. Moreover, differentiation of hESCs as embryoid bodies in serum-containing medium without the use of additional cytokines led to sequential expansion of first erythroid and then myeloid and monocytic progenitors from day 10 of culture. These cells retained the capacity to develop into formed blood elements during in vitro culture. Consistent with the development of committed hematopoietic cells, we observed the expression of transcription factors known to be critical for hematopoietic development. We thus demonstrate successful use of enforced gene expression to promote the differentiation of hESCs into a terminally differentiated tissue, thereby revealing an important role for HOXB4 in supporting their in vitro development along the hematopoietic pathway. 4 h5 U1 s: G  K0 s
      【关键词】 Human embryonic stem cells Hematopoiesis Homeobox genes
   INTRODUCTION. V$ N. a% C. l$ z" ~
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Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of embryos cultured from the blastocyst stage . If hESCs are to be clinically useful in generating HSCs, then additional strategies are needed to achieve their development and to augment the self-renewal of HSCs and their descendants, once formed./ ~& `) t5 @* A7 S! {4 d9 q+ J
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There is mounting evidence that the homeodomain transcription factor, HOXB4, has an important role in HSC self-renewal. HOXB4 has been found to be expressed in a CD34-positive human cell population enriched for HSCs and is subsequently downregulated on differentiation of these cells to committed hematopoietic progenitors . Our findings demonstrate that gain of function of this homeobox transcription factor can support development of hESCs into terminally differentiated blood elements apparently by promoting the expansion of hematopoietic progenitors and their descendant populations.
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MATERIALS AND METHODS

hESC Culture3 E$ a: M, f  A4 J: |
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The hESC line H9 was obtained from WiCell (WiCell Research Institute, Madison, WI, http://www.wicell.org). The cells were maintained in the undifferentiated state by culture on irradiated murine embryonic fibroblasts (MEFs) as described , only karyotypically normal cells at passage 45 to passage 65 were used in these experiments.
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Differentiation of hESCs as Embryoid Bodies
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The cells were incubated for 20 minutes in 1 mg/ml Collagenase IV (Gibco, Paisley, U.K., http://www.invitrogen.com) and scraped off the Petri dishes. The detached colonies were washed once in the differentiation medium (KO-D-MEM supplemented with 20% fetal bovine serum .

Generation of HOXB4-Expressing hESC Lines

HOXB4-expressing and "human recombinant" green fluorescent protein (GFP)-expressing (Stratagene, La Jolla, CA, http://www.stratagene.com) constructs were generated using the pTP6 expression vector . Three days after transfection, the cells were passed onto 60-mm gelatin-coated tissue-culture plates containing puromycin-resistant mouse fetal fibroblasts as feeders. After 3 additional days, puromycin (final concentration 1 µg/ml) was added. Puromycin-resistant colonies that appeared after approximately 12 days of selection were picked, dissociated, and plated onto 24-well gelatin-coated, feeder-containing plates, and expanded for further analysis (see below). Each clone was amplified from a single puromycin-resistant colony. Three individual HOXB4-expressing hESC clones were assessed, hereafter designated HOXB4.1, HOXB4.2, and HOXB4.3.- g) ^. I4 v9 D+ d+ d: m! R# k
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Reverse Transcription Polymerase Chain Reaction Analysis0 [  D" Q( _7 @( G

Cycle conditions were as follows: 94¡ãC for 5 minutes, then 40 cycles of 94¡ãC for 30 seconds, 60¡ãC for 30 seconds, and 72¡ãC for 30 seconds, followed by a final extension at 72¡ãC for 5 minutes. The primers used were as follows: HOXB4-untranslated region (254 base pairs ) 5'-ATC TGT CTT GTT TCC TCT GCC G-3' and 5'-TGA ATG GGC ACG AAA GAT GAG G-3', HOXB4-translated region (410 bp) 5'-TCT GTC CCC TCG GGC TCC TGC G-3' and 5'-GGC AAC TTG TGG TCT TTT TTC C-3', ß-2-microglobulin (288 bp) 5'-ACT GAA AAA GAT GAG TA T GCC TGC CGT GTG AAC C-3' and 5'-CCT GCT CAG ATA CAT CAA ACA TGG AGA CAG CAC T-3'.

Quantitative Reverse Transcription Polymerase Chain Reaction4 H1 E5 |* j( J( c# q

Total RNA was extracted using the RNeasy Mini Kit (Qiagen) which included a RNase-free DNase digestion step. Following manufacturers instructions, 0.5 µg of RNA was reverse transcribed using 200 units of Superscript II (Invitrogen) and 0.5 µg of random primers (Promega). Real-time Taqman polymerase chain reaction (PCR) was performed using an ABI 7700 with 1x Mastermix (Eurogentec, Seraing, Belgium, http://www.eurogentec.be) and 500 ng of cDNA. Primers and probes used in the real-time Taqman PCR were as follows: HOXB4, 1x Assays-on-Demand (Hs 00256884_m1; Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com); stem cell leukemia gene (SCL), 1x Assays-on-Demand (Hs00268434_m1, Applied BioSystems); GATA1, 1x Assays-on-Demand (Hs00231112_m1; Applied BioSystems); hydroxymethylbilane synthase (HMBS), forward primer GGAGCCATGTCTGGTA-ACGG, reverse primer CCACGCGAATCACTCTCATCT, probe TTTCTTCCGCCGTTGCAGCCG. Cycle conditions were as recommended by Eurogentec. The comparative threshold cycle (CT) method was used to analyze data, with HOXB4, SCL, and GATA1 expression levels calibrated to the expression of the housekeeping gene HMBS.
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Southern Blot Analysis5 J, p) K- l, M0 E

Genomic DNA (10 µg) from distinct HOXB4-transfected hESC clones was digested with EcoR1 and electrophoresed through 1% agarose gels followed by transfer to Hybond N nylon membrane (Amersham Biosciences, Chalfont St. Giles, U.K., http://www4.amershambiosciences.com). The probe used for hybridization to the Southern membrane was labeled with . After hybridization, the membrane was exposed to X-ray film for 5 days.

Immunocytochemistry

Colonies of wild-type and HOXB4-transfected hESCs cells were fixed in 4% (wt/vol) paraformaldehyde (Sigma-Aldrich, Gillingham, U.K., http://www.sigmaaldrich.com). Cells were stained with either HOXB4 (I12; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww), Oct-4 (C-10; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), or the appropriate isotype control antibodies (HOXB4, rat IgG2a ; Sigma-Aldrich). The cells were viewed in PBS by epifluorescence microscopy with the appropriate filter using a Zeiss Axiovert 200M (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Western Blot Analysis/ o8 F2 h$ c& b) R5 S
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Fifty micrograms of total protein was run on a NuPage gel (Invitrogen). Gels were blotted onto a nitrocellulose membrane (Amersham Biosciences), which was then stained with rat anti-HOXB4 antibody (Developmental Studies Hybridoma Bank) followed by secondary anti-rat Ig antibody conjugated to horseradish peroxidase (HRP) (DakoCytomation). Membranes were developed using ECL Western blotting detection system (Amersham Biosciences) as per the manufacturer¡¯s instructions. After stripping of the membrane, a mouse anti-actin antibody (Chemicon) followed by secondary goat anti-mouse antibody conjugated to HRP (DakoCytomation) was used as a positive control.1 p! B* r1 T- S# Z! W

Flow Cytometry" t6 a/ ~4 v8 y4 r4 J. Z
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A single-cell suspension was produced from hESCs cultured on MEFs or from embryoid bodies as follows. Cells were washed once in PBS before incubation at 37¡ãC in 1 mg/ml of Collagenase IV for 20 minutes. The cells were then washed once in PBS and incubated in enzyme-free cell dissociation buffer (Invitrogen) for 20 minutes at 37¡ãC. Finally, cells were gently titurated before filtering through a 40-µm mesh.& i, B; J+ e2 N& `" J8 ?  `5 j
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For flow cytometry, cells were resuspended at approximately 0.1 x 105¨C1.0 x 105 cells per ml with PBS x 3% normal goat serum containing 0.1% azide (Serotec) and stained with fluorochrome-conjugated monoclonal antibodies, CD34-fluorescein isothiocyanate (FITC), CD34-phycoerythrin (PE), CD45-PE (all from BD Biosciences), and Glycophorin A-PE (DakoCytomation) or their corresponding isotype controls. For detection of stage-specific embryonic antigen-4, cells were stained with the primary SSEA-4 monoclonal antibody (MC-813¨C70; Developmental Studies Hybridoma Bank) or IgG3 isotype control (BD Biosciences) and then stained with a FITC-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich). All cells were incubated with 20 µl/ml 7-amino-actinomycin D (7-AAD) viability dye (Immunotech, Luminy, France, http://www.immunotech.com) and live cells identified by 7-AAD exclusion were analyzed for surface-marker expression using a BD FACSCalibur flow cytometer with Cell Quest software (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). For all dot plots, gates were set with at least 99.9% of the cells stained with the isotype control in the lower left (wild-type and HOXB4-transfected hESCs) or lower left and right (GFP-transfected hESCs) quadrants.7 F4 S  a3 U" W* _/ F* U1 C

Clonogenic Assays/ S+ [! l3 D3 A  z" ^, ~
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A single-cell suspension was produced as described above. Human hematopoietic progenitor assays were performed by plating single-cell suspensions of undifferentiated hESCs and differentiated hESCs into Methocult GF  medium (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) consisting of 1% methylcellulose, 30% FBS, 1% BSA, 50 ng/ml stem cell factor, 20 ng/ml granulocyte-macrophage colony-stimulating factor, 20 ng/ml IL3, 20 ng/ml IL6, 20 ng/ml granulocyte colony-stimulating factor, and 3 units/ml erythropoietin. Cells were aliquoted in duplicate samples at between 1 x 104 and 1 x 105 cells per plate. After incubation at 37¡ãC in 5% CO2 for 15 days in a humidified atmosphere, differential colony counts were performed based on morphological characteristics. To identify specific cell types, individual colonies were aspirated from the plates, washed once in PBS, and then resuspended in PBS-1% human albumin solution before cytospin and staining with modified Wright¡¯s stain (Bayer, Newbury, U.K., http://www.bayer.co.uk) on a Hematek 1000 slide stainer (Miles Laboratories Inc.).1 _3 L$ V, Z, }1 ]1 }
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Statistical Analysis: Y5 v8 g4 ^; I$ P+ O, K

For flow cytometry and CFC data, results are expressed as mean ¡À SEM. Statistical significance was assessed using the unpaired Student¡¯s t test. Results were considered to be significantly different from control or each other if p

RESULTS% W, w; O, x, e# i/ i: i

HOXB4 Transfection of hESC

We expressed the HOXB4 cDNA in hESCs (HOXB4-hESCs) using the pTP6 vector .7 A' h5 x+ D' F8 b/ {( S

Figure 1. HOXB4 expression in WT-hESCs, control GFP-hESCs, and HOXB4-hESCs. (A): Map of the HOXB4-pTP6 and GFP-pTP6 vectors. pTP6 vector contains the CAGG promoter followed by the HOXB4 or GFP cDNA and an IRES, with puror allowing strong selection for transgene expression. (B): Semiquantitatively reverse transcription polymerase chain reaction (PCR) analysis of HOXB4-UTR and HOXB4-TR of WT-hESCs, GFP-hESCs, and HOXB4-hESCs clones (HOXB4.1, HOXB4.2, and HOXB4.3). A sample of cDNA from the erythroleukemia cell line K562 was used as a positive control, and a sample containing no cDNA (H2O) was used as a negative control. The housekeeping gene B-2-m was used to normalize the PCR. (C): Real-time PCR analysis of HOXB4 expression in WT-hESCs, GFP-hESCs, and HOXB4-hESCs (clones HOXB4.1, HOXB4.2, and HOXB4.3) on day 0 (undifferentiated). Gene expression relative to K562 was calibrated by the comparative CT method (p > .18). Data points represent the mean expression of two independent experiments ¡À SEM. (D): Southern blot analysis of HOXB4-hESCs (clones HOXB4.1, HOXB4.2, and HOXB4.3) demonstrating that each clone has a distinct integration site. The presence of two bands for HOXB4.1 suggests that two copies of the plasmid have integrated into the genome, and the presence of a single band for HOXB4.2 and HOXB4.3 suggests transfection of a single copy of the plasmid in these clones. (E): Immunocytochemistry of WT-hESCs with HOXB4 antibody and secondary fluorescein isothiocyanate (FITC)-conjugated antibody, HOXB4-hESC clone with HOXB4 antibody and secondary FITC-conjugated antibody, and HOXB4-hESCs with Oct4 antibody and secondary Cy3 antibody. Hoescht dye was used to stain the nuclei. Scale bars = 50 µm. (F): Western blot analysis of HOXB4 expression in WT-hESCs, GFP-hESCs, and HOXB4-hESCs (clones HOXB4.1, HOXB4.2, and HOXB4.3) on day 0 (undifferentiated). Protein extracted from the erythroleukemia cell line K562 was used as a positive control. (G): Analysis of the stem cell marker SSEA-4 by flow cytometry in WT-hESCs and HOXB4-hESCs as represented by the shaded histograms. The gated region is based on exclusion of 99.9% of cells of the isotype control (open histograms) and represents the mean ¡À SEM (n = 5 for wild-type-hESCs; n = 9 for HOXB4-hESCs). There was no difference in SSEA-4 expression. (H): Western blot analysis of HOXB4 expression in WT-hESCs, GFP-hESCs, and HOXB4-hESCs (clones HOXB4.1, HOXB4.2, and HOXB4.3) after 20 days of differentiation as embryoid bodies. Protein extracted from the erythroleukemia cell line K562 was used as a positive control. Actin was used to normalize the Western blot reactions. Abbreviations: B-2-m, ß-2-microglobulin; GFP, green fluorescent protein; GFP-hESC, green fluorescent protein-expressing hESC clone; hESC, human embryonic stem cell; HOXB4-hESC, HOXB4-expressing hESC clone; HOXB4-TR, HOXB4 translated region; HOXB4-UTR, HOXB4 untranslated region; IRES, internal ribosome entry site; puror, puromycin resistance gene; WT, wild-type.
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Lipofection using the pTP6 vector was effective in generating stable expression of the HOXB4 transgene. Following transfection, the cell lines were maintained in their undifferentiated state on MEFs. Neither untransfected wild-type hESCs (WT-hESCs) nor control GFP-hESCs expressed HOXB4. Reverse transcription-PCR of WT-hESCs, GFP-hESCs, and HOXB4-hESCs showed that gene expression was confined to the HOXB4-transfected clones (Fig. 1B). As only the translated region of the HOXB4 gene was transfected in the expression vector, we were able to determine that the HOXB4 transcription in the HOXB4-hESCs was a result of transgene expression and not endogenous expression of HOXB4 by using two sets of primer pairs, one within the translated region and another in the untranslated region of the HOXB4 gene (Fig. 1B). Real-time quantitative PCR using human HOXB4-specific primers demonstrated levels of HOXB4 transcript in the HOXB4-transfected hESCs that were generally similar to those of the erythroleukemia cell line K562 (Fig. 1C). Southern blot analysis confirmed that transgene integration sites in the selected clones were distinct from each other (Fig. 1D). Immunocytochemistry and Western blot analysis confirmed that HOXB4 expression was confined to the HOXB4-hESCs (Fig. 1E, 1F). Furthermore, immunocytochemistry confirmed that the HOXB4 protein was localized in the nucleus and that all cells within each of the colonies expressed similar levels of the transgene. Both undifferentiated WT-hESCs (data not shown) and HOXB4-hESCs similarly expressed the hESC markers Oct-4  by flow cytometry (Fig. 1G). Expression of HOXB4, Oct-4, and SSEA-4 in HOXB4-hESCs was stable for at least 15 passages (the maximum stage studied here) following transfection. To establish whether HOXB4 expression was maintained upon differentiation of HOXB4-hESC clones, Western blot analysis of EBs at day 20 of culture in serum-containing medium was carried out, confirming that HOXB4 expression was sustained and not downregulated significantly (Fig. 1H). Therefore, we considered HOXB4 transfection and gene expression to be compatible both with the undifferentiated state of hESCs and with their subsequent differentiation.

Control hESCs Exhibit a Low Level of Hematopoietic Differentiation upon Differentiation as EBs
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The experimental approach used here to examine the initial phenotypic consequences of HOXB4 gain of function was to differentiate hESCs as EBs in serum-containing medium (without additional hemogenic cytokines) during a 20-day period, analyzing the samples at 5-day intervals using flow cytometry. With this approach, differentiation of WT-hESCs and GFP-hESCs generated low levels of cells expressing hematopoietic markers. Flow cytometry identified the development of a small population of CD45-positive cells that peaked on day 20 of the experiment in both untransfected (WT-hESCs 0.6% ¡À 0.1%, both CD34-positive and CD34-negative combined; five independent experiments; Fig. 2A) and GFP-transfected (0.7% ¡À 0.1%; six independent experiments; Fig. 2B) hESCs. In WT-hESCs, CD34/CD45 dual-positive cells were less than or equal to 0.1% on all days of the experiment. Glycophorin A, a surface marker specific to erythroid cells, was not greater than 0.1% at any time point in non-HOXB4-transfected hESCs. There was not a significant difference in expression of CD34, CD45, and Glycophorin A between WT-hESCs and GFP-hESCs, both of which generated very modest numbers of hematopoietic cells." j9 g9 u7 d( Z. {

Figure 2. HOXB4-transfected hESCs generate greater numbers of cells expressing hematopoietic cell surface markers than do wild-type and control hESCs. Embryoid bodies were formed and differentiated in a serum-containing medium without the addition of further cytokines. Samples were analyzed by flow cytometry 10, 15, and 20 days after the generation of the EBs for cell surface marker expression of CD34, CD45, and Glycophorin A (arrows). (A, B): Un-transfected (WT-hESCs) (A) and GFP-transfected (GFP-hESCs) (B) hESCs rarely generate cells expressing hematopoietic cell surface markers, showing less than 1% CD45- or Glycophorin A-expressing cells. (C¨CE): Three HOXB4-transfected hESC clones (HOXB4.1-hESC, HOXB4.2-hESC, and HOXB4.3-hESC) show a significant increase in the number of hematopoietic cells generated. Quadrant percentages represent the mean ¡À SEM for five (WT-hESCs), six (GFP-hESCs), three (HOXB4.1-hESCs), six (HOXB4.2-hESCs), and six (HOXB4.3-hESCs) independent experiments. Abbreviations: hESC, human embryonic stem cell; HOXB4 hESC, HOXB4-transfected hESC clone; GFP, green fluorescent protein.. x7 B3 w4 ?' j( s

HOXB4 Augments the Generation of Hematopoietic Cells from hESCs  H( [5 _" |9 Q/ a' Z
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Three individual HOXB4-hESC clones were examined extensively (HOXB4.1-hESCs, three independent experiments; HOXB4.2-hESCs, six independent experiments; HOXB4.3-hESCs, six independent experiments). Differentiation of HOXB4-hESCs over 15 days resulted in a similar number of cells per well compared with WT-hESCs and GFP-hESCs (data not shown). Flow cytometry showed substantial increases in CD45, CD34, and Glycophorin A expression in all three clones, compared with GFP-transfected and untransfected hESC controls (Fig. 2C¨C2E). On day 20, CD45 was expressed by a total of 4.2% ¡À 0.3% of HOXB4.1-hESCs (p   C0 {6 c3 @. ]9 Y8 ~7 J

To test whether enforced HOXB4 expression is additive or simply a substitute for the effects that can be achieved with cytokines, factors at concentrations previously shown to support hematopoietic development from hESCs were added to the differentiation medium as described .

Figure 3. Cytokines augment hematopoietic development from HOXB4-hESCs. Proportion of CD45  cells following 15 days (A) and 20 days (B) of in vitro differentiation as EBs from HOXB4-hESCs without (HOXB4/no cytokines) and with (HOXB4/plus cytokines) additional cytokines in the medium relative to control EBs not treated with cytokines (control/no cytokines). On day 15, HOXB4 resulted in a 15-fold increase in CD45  cells compared with control, with an additional threefold further increase on addition of cytokines (*, t test comparing HOXB4 without additional cytokines and HOXB4 with additional cytokines, p
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In parallel to flow cytometry, we also analyzed samples by Q-PCR for SCL/Tal-1 (hereafter called SCL) and GATA-1 expression in the absence of added cytokines. SCL plays a crucial role in hematopoietic development, as evidenced by both the SCL knockout mouse, which is an early lethal as a result of failure of embryonic blood development . Time course analysis by Q-PCR of control GFP-hESCs suggested a trend of slightly increased expression of SCL and GATA-1 (Fig. 4A and 4B, solid lines). Compared with GFP-hESCs, however, HOXB4-hESCs showed highly significant increases in SCL (p = .016) and GATA-1 (p = .0005) expression when they were differentiated as EBs (Fig. 4A and 4B, interrupted lines). These pronounced increases in expression of SCL and GATA1 by HOXB4-hESCs progressed over time (SCL, p = .0011; GATA1, p = .0005), first becoming apparent on day 10. The maximum increase of expression for both genes (observed on day 20 of the experiment) was approximately 90-fold for SCL and 350-fold for GATA1. Consequently, HOXB4 expression is capable of amplifying a subset of cells that emerges between day 5 and day 10 and results in increased expression of SCL and GATA-1, two representative genes critical to the pathway of hematopoietic development.$ r# w& w4 }0 G- P( K

Figure 4. HOXB4-expressing human embryonic stem cells (hESCs) show upregulation of genes associated with hematopoiesis upon differentiation. Quantitative real-time PCR analysis of SCL (A) and GATA1 (B) expression by GFP-hESCs (solid lines) and HOXB4.2-hESCs (interrupted lines) differentiated in serum containing medium without the addition of further cytokines over 20 days. Gene expression relative to undifferentiated hESCs was calibrated by the comparative CT method. Data points represent the mean expression of two independent experiments ¡À SEM." k' K5 q& ]0 j& ?7 Z" i, ^
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HOXB4 Expression Enhances Clonogenic Hematopoietic Progenitor Development from hESCs
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To assess the consequences of HOXB4 expression for in vitro hematopoietic development, we carried out a clonogenic progenitor assay. This was accomplished by determining the number of hematopoietic colonies formed by dissociating and subculturing EBs at different stages of their development (0¨C20 days; Materials and Methods). At the earliest time points (0 and 5 days of culture), there was no significant increase in the total number of colonies (including all hematopoietic cell lineages) formed from the HOXB4-hESC clones, resembling the paucity of such colonies in cultures of wild-type and GFP-hESCs. On day 10 of differentiation, however, there were significant increases in the mean numbers of total colonies from HOXB4-transfected hESCs compared with GFP-hESCs as follows (Fig. 5A¨C5C): HOXB4.1-hESCs, 83 ¡À 15 colonies per 106 cells plated (p 1 }& {5 H0 o/ w
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Figure 5. HOXB4 expression in human embryonic stem cells induces increased hematopoietic progenitors. (A¨CC): Total number of hematopoietic colony-forming progenitor cells formed from WT-hESCs, GFP-hESCs, and HOXB4-transfected (HOXB4.1-hESCs, HOXB4.2-hESCs, and HOXB4.3-hESCs) human embryonic stem cells differentiated as embryoid bodies in serum-containing medium after 10, 15, and 20 days of embryoid body differentiation. Error bars represent SEM for three to five independent experiments (*, p

Interestingly, the composition of the hematopoietic colonies formed from HOXB4-expressing hESCs varied with the stage of EB development, as well as between hESC sublines. In all three HOXB4-expressing clones, erythroid colony (burst forming unit-erythroid ) colony development peaked on day 15 or 20. Although all three HOXB4-hESCs clones produced significantly more erythroid, myeloid, and monocytic colonies than either WT-hESCs or GFP-hESCs, there was a noticeable difference in overall colony-forming efficiency between the three HOXB4-hESCs clones analyzed (Fig. 5A¨C5H). There were, however, general similarities in the patterns of the different blood lineages formed, with a preponderance of myeloid (CFU-GM) and monocytic (CFU-M) colony formation, particularly at the later stages of EB culture (days 15 and 20). An exception was that colony forming unit-granulocytic, erythroid, monocytic (CFU-GEM) colonies, representing the most primitive of hematopoietic myeloid progenitors, were only observed at a frequency of 10 colonies per 106 cells plated using HOXB4.3-hESCs. No erythroid colonies (BFU-E) and no multipotent colonies (CFU-GEM) were detected in any of 11 individual experiments on WT-hESCs and GFP-hESCs.
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Finally, hematopoietic cells arising from HOXB4-hESCs were capable of developing into fully formed blood elements. Colonies scored as BFU-E (Fig. 5I) contained cells of morphologically maturing erythroid lineage with characteristic nuclei, eosinophilic cytoplasm, and occasional anucleate cells (Fig. 5J). Colonies scored as CFU-GM (Fig. 5K) contained cells comprising all morphological stages of myeloid maturation; blast, pro-myelocyte, myelocyte, and metamyelocyte through to segmented forms with three or more nuclear segments. Within the CFU-GM, eosinophils and their precursors, as well as the occasional monocytoid cell, were seen (Fig. 5L). Colonies scored as CFU-M (Fig. 5M) contained exclusively lipid-laden monocytoid cells (Fig. 5N). Colonies scored as CFU-GEM contained cells from all lineages. Thus, HOXB4 expression not only increased the total number of hematopoietic colonies but was also consistent with their development in vitro into terminally differentiated erythroid or myeloid cells.
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DISCUSSION" d- n; n7 x' w! }* v/ Y# _
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This report demonstrates the use of transcription factor overexpression to promote the development of a clinically useful tissue from hESCs. Stable HOXB4 expression in hESCs was compatible with the undifferentiated state (as evidenced by Oct4 and SSEA-4 expression), thus permitting prolonged passaging of individual hESC clones. Moreover, differentiation of these hESC clones in a serum-containing medium without additional cytokines resulted in a significant increase in hematopoietic development. Addition of cytokines further increased the yield of hematopoietic cells by HOXB4-expressing hESCs, suggesting that HOXB4 may act independently to enhance such development. Our findings thus extend to hESCs the utility of homeodomain transcription factors as a means of augmenting in vitro hematopoietic development, as previously demonstrated for mouse ESCs .
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HOXB4 overexpression in hESCs resulted in sequential increases in erythroid cells and leucocytes, as evidenced by increased expression of Glycophorin A . Thus, HOXB4 expression clearly promotes the hematopoietic development in the descendants of hESCs undergoing differentiation as EBs even in the absence of animal stromal cells and further cytokine additives.

In a recent study, HSCs derived from hESCs have been shown to be capable of repopulating hematopoiesis in primary and secondary recipient sheep . Such HOXB4-expressing cells may ultimately be capable of significantly increasing reconstitution of multilineage hematopoiesis in animal models.
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Similar experiments overexpressing HOXB4 in murine ES cells resulted in increases only in erythroid and mixed erythroid/myeloid colonies and not in granulocytic or monocytic colonies . Therefore, the timing of expression, level of expression, and cellular context may all affect the outcome of HOXB4 expression and may thus account for different hematopoietic phenotypes observed when overexpressing HOXB4 using different cells or methods.% d( p7 Y! L$ q3 [8 l$ E7 v
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A similar explanation might account for the marked differences in overall colony-forming efficiency between HOXB4-expressing hESC lines. The HOXB4 hESC cell lines were initially selected for their ability to maintain transgene expression in the undifferentiated state. These HOXB4-expressing hESCs clones were shown by real-time PCR, Western blotting, and immunocytochemistry to have transgene expression levels generally similar to each other and to the erythroleukemia cell line K562. Nevertheless, the HOXB4-hESC clones differed significantly from each other in hematopoietic output, suggesting that other variables could also affect ultimate phenotypic consequences during the course of in vitro differentiation to hemogenic progenitors.
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Even with constitutive expression of HOXB4 mRNA and protein for multiple generations in the undifferentiated state and for up to 20 days of EB development, only a fraction of such transgenic hESCs undergo hematopoietic differentiation. This raises the question of what limits the incidence of hematopoietic development in such cultures. It seems unlikely that HOXB4 acts directly to promote the differentiation of hESCs into HSCs. A more probable mechanism of HOXB4 action in hESCs is that it affects hematopoietic cells that have spontaneously differentiated from hESCs to mesoderm and thence to HSCs. Such a mechanism would be consistent with HOXB4 effects in mouse HSCs derived from ESCs and other sources, where it is thought to act by amplifying the number of HSCs and their descendants through an increase in HSC self-renewal . In any case, hESC-based generation of hematopoietic cells for clinical application would require a substantial improvement in the efficiency of the differentiation steps leading to HSC formation.$ A) S% ^/ i& w1 T  p

CONCLUSION9 S% F4 [' R) O9 x. N' J. N
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By demonstrating that HOXB4 can promote hematopoietic development from hESCs, this work provides a model for amplification of differentiated cell types from hESCs with potential therapeutic application. It thereby addresses a problem that must be solved in any attempt to provide clinically useful tissues, namely self-renewal of progenitor and intermediate (transit amplifying) stem cell populations to generate sufficient numbers of terminally differentiated cells. The remaining challenge of establishing substantial long-term, multilineage human hematopoiesis in an animal HSC transplant model should be facilitated by such an approach.
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ACKNOWLEDGMENTS0 x/ A* ?6 I& a2 _  b
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We are grateful for the help received from members of the Departments of Surgery, Pathology and Haematology at the University of Cambridge, in particular Professors Andrew Bradley, Tony Green, and Charles ffrench-Constant for support during this work and Dr. Isabelle Bouhon for encouragement during its inception. We thank Dr. T. Pratt (University of Edinburgh, Edinburgh, U.K.) for the pTP6 vector, Dr. R.K. Humphries (British Columbia Cancer Agency, Vancouver, BC, Canada) for the HOXB4 cDNA, and Dr. K. Vintersten (Samuel Lunenfeld Research Institute, Toronto, ON, Canada) for the puromycin resistant feeders. We are also grateful to Dr Mickie Bhatia (University of Western Ontario, London, ON, Canada) and his team for advice with respect to analysis of hESC-derived hematopoietic cells. This study was supported by a Wellcome Trust Clinical Research Training Fellowship (K.M.B.), a Raymond and Beverly Sackler Studentship (K.M.B.), a Medical Research Council International Appointments Initiative grant (L.V., M.R.J.A., R.A.P.), a Human Frontiers Science Program grant (RG0110/2000-M; R.A.P., L.V.), and a Cambridge-MIT Institute grant (J.R.S.)." h% {* c( y+ p$ T* p* K

DISCLOSURES

R.A.P. has a financial interest in Stemnion, LLC.
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