First published online April 24, 2008
Stem Cells
Vol. 26 No.
6
June 2008, pp.
1658
-1662
doi:10.1634/stemcells.2007-0952; www.StemCells.com
© 2008 AlphaMed Press
Impairment of Granulo-Monocytic Development of Human Common Myeloid Progenitors but Not of Granulo-Monocytic Progenitors by Decreasing Stem Cell Leukemia/T-Cell Acute Leukemia 1 Expression
Philippe Brunet de la Grangea,b,
Estelle Zinka,b,
Florence Armstronga,b,
Marie-Christine Rouyeza,b,
Françoise Pflumioa,b
aInstitut Cochin, Université Paris Descartes, Paris, France;
bINSERM, Paris, France
Key Words. Regulation of hematopoiesis • Myeloid development • CMP • GMP • TAL1 expression
Correspondence:
Correspondence: Philippe Brunet de la Grange, Ph.D., Département d'Hématologie, Institut Cochin, U567 INSERM, CNRS UMR 8,104, Université Paris 5, Faculté de Médecine René Descartes UM 3, 123 Bd de Port Royal, Paris, 75,014, France. Telephone: 33-1-46-54-77-04; Fax: 33-1-46-54-91-38; e-mail: philippe.brunet-de-la-grange{at}cea.fr
Received on November 14, 2007;
accepted for publication on April 10, 2008.
First published online in STEM CELLS EXPRESS April 24, 2008.
 |
ABSTRACT
|
|---|
We recently showed that Stem Cell Leukemia/T-cell Acute Leukemia 1 (SCL/TAL1) regulates hematopoiesis from hematopoietic stem cells to committed myeloid progenitors compartment. However, in this heterogeneous compartment, the precise role of TAL1, that is largely debated, remains to be clearly defined, notably at the common myeloid progenitor (CMP) and granulo-monocytic progenitor (GMP) levels. Using small hairpin (sh)RNA lentiviral constructs, we decreased TAL1 expression in sorted human CMP and GMP subpopulations that were then assayed for erythroid and granulo-monocytic (GM) differentiation. Decreased TAL1 expression in CMP resulted in rare erythroid colonies, in a 2–3 fold reduction of GM colony number in clonogenic assays and in a 3.6–5.6 decreased production of CD14+CD15+ GM cells in liquid culture. Moreover, analysis of transcript profile of gene involved in GM differentiation showed that GM cells expressing shRNA-TAL1 construct displayed decreased levels of g-csfr, c/ebp
, and mpo and high levels of gata-2 transcripts, indicating a blocking of GM differentiation. In contrast, GM differentiation of GMP remained unaffected when TAL1 transcript levels were decreased. These data definitively delineate the human myeloid progenitors that are regulated by TAL1.
Disclosure of potential conflicts of interest is found at the end of this article.
|
INTRODUCTION
|
|---|
Stem Cell Leukemia/T-cell Acute Leukemia 1 (TAL1) is a basic helix-loop-helix transcription factor essentially known for its active role in stem cells emergence in mouse embryo and also in mouse and human adult erythropoiesis and megakaryopoiesis [1–3]. TAL1 function in other hematopoietic compartments was recently refined [4–7], and we have shown that decreased TAL1 expression in human cord blood CD34+ cells strongly interferes with hematopoietic development from stem cells to committed progenitors. However the precise role of TAL1 in the hierarchy of myeloid progenitors remains quite elusive [3, 7, 8]. In mice, conditional knockout experiments did not show any effect of TAL1 in committed myeloid progenitors; only overexpression of TAL1 led to moderate/good increase of myeloid progenitors such as colony forming cells (CFC) [7, 8]. In human studies, except for mega-erythroid (ME) progenitors, the role of TAL1 was poorly documented. In our previous studies [6, 9], we showed that over-expression and inhibition of TAL1 interfere with human hematopoietic stem cells development, and we showed for the first time that TAL1 inhibition impaired the development of granulo-monocytic (GM) cells from committed myeloid progenitors. However, the question of where TAL1 acts in this heterogeneous compartment was not addressed so far. Committed myeloid progenitors consist mainly in three subpopulations: (1) CMP (Common Myeloid Progenitors) possess a GM and ME potential, (2) GMP and (3) MEP (Mega-Erythroid Progenitors) both derive from CMP and exhibit respectively restricted GM and ME potentials. Recently, Zhang et al. showed that tal1 transcripts are present in CMP and GMP [10]. In the present work, we decreased tal1 mRNAs expression in enriched CMP and GMP fractions and assayed these progenitors for erythroid and/or GM differentiation. Our results show that decreased tal1 expression in CMP prevents the development of erythroid and GM colonies in semi-solid cultures and the production of GM cells in liquid culture. By contrast, GMP differentiation remained unaffected. Moreover, the transcript profiles of genes usually involved in GM differentiation was only affected in CMP. These data provide important new data about the role of TAL1 in human myeloid progenitors, establishing the last milestone of TAL1 spectra of action during human hematopoiesis.
|
MATERIALS AND METHODS
|
|---|
Lentiviral Constructs
shRNA-TAL1 and shRNA-Ctrl constructs were designed and production of lentiviral vectors was performed as previously described [2, 9].
Cell Sorting and Transduction
Cord blood (CB) samples were collected from healthy infants with the informed consent of the mothers, according to approved institutional guidelines. CD34+ cells were purified by immunomagnetic selection (StemCell Technologies, Vancouver, BC, CA, http://www.stemcell.com).
CD34+Lin– cells were sorted after labeling with monoclonal antibodies (MoAbs) CD34-FITC and PE conjugated CD4, CD7, CD8, CD56, CD19, CD127, CD14, CD15, Glycophorin-A, CD42b (Beckman Coulter, Villepinte, France, http://www.beckmancoulter.com).
CD34+Lin– cells were transduced with shRNA-TAL1 or shRNA-Ctrl lentiviral vectors as described.
Transduced CD34+ cells were then labeled with CD45RA-PE (Beckman Coulter) and CD123-PC5 (Becton Dickinson, Le Pont-de-Claix, France, http://www.bd.com) and GFP+CD123+CD45RA– and GFP+CD123+CD45RA+ were sorted as described [10] (Fig. 1A, top) with a purity at least of 90% (Fig. 1A, bottom).
View larger version (30K):
[in this window]
[in a new window]
|
Figure 1. Purification of human hematopoietic cells enriched for CMP and GMP potentials. CD34+ cells were immuno-magnetically purified from ficolled umbilical cord blood. They were then labeled with CD34-FITC and Lin-PE antibodies and sorted for CD34+/Lin– cells. CD34+/Lin– cells were plated 3 days for transduction with shRNA-TAL1 or shRNA-Ctrl lentiviral constructs. (A): Cells were labeled and analyzed for CD123 and CD45RA expression in the GFP+ fraction (transduced cells) (upper panel) and GFP+CD123+CD45RA–CD34+ and GFP+CD123+CD45RA+CD34+ were sorted and analyzed for purity (bottom). (B): Hematopoietic potentials exhibited by CD123+CD45RA–CD34+ and CD123+CD45RA+CD34+ cells transduced with the shRNA-Ctrl lentiviral constructs and tested in CFC assay (top) and in single liquid culture allowing B lymphoid, NK and GM differentiation (bottom).
|
|
Cell sortings were performed with an EPICS Beckman Coulter Cell Sorter.
Cultures
B lymphoid/Natural killer/Granulo-monocytic (B/NK/GM) cultures.
Transduced CD123+CD45RA+CD34+ and CD123+CD45RA–CD34+ cells (4.104/mL) were plated in a single culture for 3 weeks on the stromal cell line mouse stromal 5 in IMDM 3% fetal calf serum 6,150 (StemCell Technologies), stem cell factor (SCF) (50 ng/mL; Amgen, Neuilly sur Seine, France, http://www.amgen.fr), IL15 (1 ng/mL; StemCell Technologies) and analyzed for B lymphocytes, NK and GM differentiation by flow cytometry.
CFC assays.
Duplicates of 300 CD123+CD45RA–CD34+ and CD123+CD45RA+CD34+ sorted cells were seeded in methylcellulose as described.
GM liquid culture.
For specific GM differentiation CD123+ CD45RA–CD34+ and CD123+CD45RA+CD34+ sorted cells (2.104/mL) were cocultured on MS-5 cells in IMDM 15% BIT9500 (StemCell Technologies), IL3 (10 ng/mL; Diaclone, France), SCF (10 ng/mL), Flt3 ligand (50 ng/mL; Diaclone, Besançon, France, http://www.diaclone.com) and G-CSF (15 ng/mL; Amgen) during 3 weeks. Proliferation and differentiation of GFP+ cells were evaluated at 7, 14 and 21 days by flow cytometry.
Flow cytometry.
Lineage analysis of cultured cells was performed on a FACSCalibur (Becton Dickinson) using PE, PC5 or APC conjugated mouse anti-human MoAbs: CD45, CD34, CD14, CD15, CD19, CD56 (Beckman Coulter).
Real Time Quantitative PCR
cDNA were generated from extracted RNAs of 105 cells collected at different time points of liquid culture and were then used for quantitative polymerase chain reaction analysis (Sybr Green, Roche Diagnostic, Melan, France, http://www.roche.com). Sequences of oligonucleotides are available on request.
Statistics.
Statistical analysis was performed using the paired Student t-test.
|
RESULTS
|
|---|
Transduction Does Not Affect the Potential of Sorted CD123+CD45RA–CD34+ and CD123+CD45RA+CD34+ Hematopoietic Cells
In the first step of the experiment, we evaluated whether sorted transduced CD123+CD45RA–CD34+ and CD123+CD45RA+ CD34+ cell populations had similar functional characteristics as the originally described CMP and GMP since compared to the original procedure, our investigation required a 3-day transduction period of CD34+Lin– cells. In CFC assay, CD123+CD45RA–CD34+ transduced cells produced a similar numbers of erythroid and GM colonies (Fig. 1B, upper panel; 45.9 ± 11.5 burst-forming unit-erythroid (BFU-E) vs. 59 ± 18.2 colony-forming unit-GM (CFU-GM) per 300 cells; p > .05) whereas CD123+CD45RA+CD34+ transduced cells were highly enriched for GM potential (17.2 ± 16.5 BFU-E vs. 78 ± 32.9 CFU-GM; n= 6, p= .001). Here, the remaining erythroid colonies might be attributed to the 10% of CD123+CD45RA–CD34+ cells that contaminated the CD123+CD45RA+CD34+ fraction after cell sorting (Fig. 1A, bottom). Importantly, the GM colonies generated from CD123+CD45RA+CD34+ contained fewer cells (a mean of 7,256 cells/CFU-GM) than CD123+CD45RA–CD34+ derived colonies (63,676 cells/CFU-GM, Fig. 1B, top) showing a lesser proliferating capacity, arguing for a more differentiated state and an absence of immature highly proliferating progenitors. Since CD45RA was shown to be expressed on lymphoid progenitors [11, 12] we then tested whether the CD123+CD45RA+CD34+ transduced cells contained lymphoid potentials. Sorted cells were cultured in B/NK/GM conditions in 3-week short-term cultures that characterize the potential of committed progenitors. As expected from the Lin– depletion, CD123+CD45RA+CD34+ transduced cells cultured in B/NK/GM conditions produced 70% of CD14+CD15+ cells (GM cells) and less than 15% of B or NK cells (Fig. 1B, bottom). Importantly, the CD123+CD45RA–CD34+ fraction tested in the same conditions were even more deprived of B or NK cell potentials. Taken together, these results show that despite of remaining B/NK potentials in the CD123+CD45RA+CD34+ fraction, sorting of transduced cells according to CD123 and CD45RA expression allowed an efficient enrichment in CMP (CD123+CD45RA–CD34+) and in GMP (CD123+CD45RA+CD34+) progenitors.
SCL/TAL1 Is Essential for E/GM Development from CMP but Not for GM Development from GMP
Small hairpin (sh)RNA-TAL1 transduced CMP and GMP-enriched cells were assayed for their clonogenic potential in methylcellulose (CFC assays) and for GM differentiation in short-term liquid culture that mainly address committed progenitors. Results show that decreased tal1 mRNA expression led to an eight-time (50.6 ± 9.7 vs. 8.6 ± 7.3; p= .0002) decrease of erythroid colonies (BFU-E) as expected. Importantly we also observed a significant decrease (59.3 ± 23.4 vs. 25.9 ± 14.6; p= .026) of the number of GM colonies (CFU-GM) produced from the CMP fraction (Fig. 2A). By contrast the number of CFU-GM generated from shRNA-TAL1 GMP cells was unaffected indicating that, in contrary to CMP, TAL1 is not required for proliferation and differentiation of clonogenic GMP. In specific GM liquid culture of CMP, a twofold decrease of the proportion CD14+CD15+ cells in shRNA-TAL1 expressing cells was observed (Fig. 2B(i), day 14). This decrease appeared after 7 days of culture (D14: 52.8% ± 6.5 vs. 27.9% ± 1.8; p= .002) and remained significant until 21 days (83.7% ± 8.3 vs. 59.1% ± 1.5; p= .027) (Fig. 2B(ii)). Moreover, the total number of GFP+CD14+CD15+ cells produced from shRNA-TAL1 CMP was 5.6 and 3.7 times lower at day 14 (D14; p= .009) and day 21 (D21; p= .022) of culture respectively when compared to controls (Fig. 2B(iii)). Here again, decreasing TAL1 expression in GMP did not induce any significant effect on the production of GM cells in liquid culture (Fig. 2C).
Despite the fact that tal1 mRNA expression in CMP and GMP has been recently described [10], nothing is known about its expression during GM differentiation. We thus investigated the expression level of tal1 mRNA during GM liquid culture. Both CMP and GMP enriched cells contained tal1 transcripts [10]. However higher levels were detected in freshly isolated (D0)CMP as well as during culture compared to GMP. Indeed, tal1 mRNA levels were 2.7-fold less expressed in D0 GMP versus CMP and decreased rapidly during culture (Fig. 3A). Interestingly and according to the requirement of TAL1 in GM development of CMP, tal1 mRNA levels increased up to 2.25 times at the beginning of the CMP culture (Fig. 3, top) before decreasing to reach GMP values at day 14 culture time. Importantly, shRNA-TAL1 vectors modified tal1 mRNAs (Fig. 3A) and protein (data not shown) levels in both CMP and GMP (respectively 65% and 57% of decreased mRNA levels at D0; Fig. 3A) whatever culture time tested confirming the efficiency of shRNA-TAL1 lentiviral construct to diminish tal1 expression levels [9].
View larger version (28K):
[in this window]
[in a new window]
|
Figure 3. Expression of tal1 and myeloid factors in transduced CMP and GMP during GM differentiation. (A): Quantitative analysis of tal1 transcript levels during GM culture of transduced CMP and GMP. Values for CMP and GMP are relative to the D0 tal1 mRNA level of CMP in shRNA-Ctrl condition (100%). (B): mpo, c/ebp, g-csfr and gata-2 mRNAs expression levels. Values for CMP and GMP are relative (for each gene) to the mRNA level of D0 CMP in shRNA-Ctrl condition (100%). Shown are the mean of two dosages from one representative experiment out of 3. Results were normalized with the β2microglobulin (β2m) gene in each sample.
|
|
Finally, we questioned the impact of tal1 decreased expression on transcript levels of genes usually associated with granulocytic and/or monocytic differentiation. Quantitative analysis of the mRNA levels revealed a twofold decrease of c/ebp and g-csf-receptor mRNAs, and a fourfold decrease of the granulocytic marker mpo mRNA in CMP (Fig. 3B) indicating a blockage of the GM differentiation. By contrast the gata-2 mRNAs level remained 5.3 higher in shRNA-TAL1 CMP. This high gata-2 expression probably reflects the blocking of differentiation of shRNA-TAL1 cells compared to control cells in which normal differentiation occurs and is accompanied by a decrease of gata-2 mRNAs as described [13–15]. Interestingly tal1 mRNA decreased expression in GMP did not affect the expression of any of the transcripts studied correlating with the absence of effect on GM differentiation.
|
DISCUSSION
|
|---|
Taken together, our data confirm a role for TAL1 at the committed myeloid progenitors level [7, 9] and clearly precise a selective role for TAL1 in GM development of CMP but not of GMP. These data that contrast with those of mouse conditional knockout models [3] refine our previous work and clearly delineate the human myeloid progenitors that are regulated by TAL1 (Fig. 4). The discrepancy with previously reported data on tal1 conditional knock-out mouse model can be explained by the difference in cell origin (mouse vs. human), and/or the type of cell investigated (adult bone marrow cells in the knock-out mouse model vs. post-natal cord blood cell in our study). Moreover and different from our data, in the knockout mouse model, tal1 was completely extinguished and the total absence of TAL1 protein may lead to compensatory mechanisms in myeloid cells also, as previously suggested by Hall et al. for the erythroid differentiation [16]. These results underline the importance of tal1 expression levels in regulating hematopoiesis. Indeed, Curtis et al [5] previously showed selective effects due to haploinsufficiency of tal1 in the mouse model as also described for other transcription factors such as GATA-2 [17]. Together with the level of tal1 expression, the relative proportion of TAL1 isoforms (truncated and full-length) generated by translational and transcriptional regulation, has also been reported to be important in regulating hematopoiesis notably at the lineage choice level between eythroid and megakaryocytic development [18–19]. These points remain to be explored notably during the commitment of CMP towards GMP or MEP.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 4. Recapitulative schema of TAL1 function in adult human hematopoiesis. Black lines and bold characters represent the differentiation pathways where TAL1 is involved, gray dotted lines and slim characters represent differentiation pathways where TAL1 is useless.
|
|
|
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
|
|---|
The authors indicate no potential conflicts of interest.
|
ACKNOWLEDGMENTS
|
|---|
We acknowledge P.H. Roméo and B. Gerby for helpful discussion and critical reading of the manuscript. We acknowledge S. Kusy for Western blot analysis. We are grateful to the midwives of Clinique des Noriets, Vitry-sur-Seine, for sampling cord bloods.
P.B.G. is supported by a fellowship from Agence Nationale pour la Recherche (ANR, NT05-2-44232), E.Z. was a fellow from CHU de Nancy (France), F.A. is supported by a fellow from Ligue Nationale Contre le Cancer (LNCC). The project is supported by grants from LNCC (RAB05013KKA), INSERM (ASE04144/AMA03019) and ANR.
|
FOOTNOTES
|
|---|
Author contributions: P.B.G., E.Z., F.A. and M.C.R. and F.P.: performed research and analyzed data; P.B.G. and F.P. wrote the paper.
|
REFERENCES
|
|---|
- Valtieri M, Tocci A, Gabbianelli M et al. Enforced TAL-1 expression stimulates primitive, erythroid and megakaryocytic progenitors but blocks the granulopoietic differentiation program. Cancer Research 1998;58:562–569.[Abstract/Free Full Text]
- Ravet E, Reynaud D, Titeux M et al. Characterization of DNA-binding-dependent and -independent functions of SCL/TAL1 during human erythropoiesis. Blood 2004;103:3326–3335.[Abstract/Free Full Text]
- Hall MA, Curtis DJ, Metcalf D et al. The critical regulator of embryonic hematopoiesis, SCL, Is Vital In The Adult For megakaryopoiesis, erythropoiesis, And Lineage Choice in CFU-S12. Proceedings Of the National Academy of Sciences of the United States of America 2003;100:992-997.
- Mikkola HK, Klintman J, Yang H et al. Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene. Nature 2003;421:547–551.[CrossRef][Medline]
- Curtis DJ, Hall MA, Van Stekelenburg LJ et al. SCL is required for normal function of short-term repopulating hematopoietic stem cells. Blood 2004;103:3342–3348.[Abstract/Free Full Text]
- Reynaud D, Ravet E, Titeux M et al. SCL/TAL1 expression level regulates human hematopoietic stem cell self-renewal and engraftment. Blood 2005;106:2318–2328.[Abstract/Free Full Text]
- Kunisato A, Chiba S, Saito T et al. Stem cell leukemia protein directs hematopoietic stem cell fate. Blood 2004;103:3336–3341.[Abstract/Free Full Text]
- Goardon N, Schuh A, Hajar I et al. Ectopic expression of TAL-1 protein in Ly-6E. 1-htal-1 transgenic mice induces defects in B- and T-lymphoid differentiation. Blood 2002;100:491–500.[Abstract/Free Full Text]
- Brunet de la Grange P, Armstrong F, Duval V et al. Low SCL/TAL1 expression reveals its major role in adult hematopoietic myeloid progenitors and stem cells. Blood 2006;108:2998–3004.[Abstract/Free Full Text]
- Zhang Y, Payne KJ, Zhu Y et al. SCL expression at critical points in human hematopoietic lineage commitment. STEM CELLS (Dayton, Ohio) 2005;23:852–860.
- Manz MG, Miyamoto T, Akashi K et al. Prospective isolation of human clonogenic common myeloid progenitors. Proceedings Of The National Academy of Sciences of the United States of America 2002;99:11872-11877.
- Huelin C, Gonzalez M, Pedrinaci S et al. Distribution of the CD45R antigen in the maturation of lymphoid and myeloid series: the CD45R negative phenotype is a constant finding in T CD4 positive lymphoproliferative disorders. British Journal Of Haematology 1988;69:173–179.[CrossRef][Medline]
- Akashi K, Traver D, Miyamoto T et al. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 2000;404:193–197.[CrossRef][Medline]
- Kumano K, Chiba S, Shimizu K et al. Notch1 inhibits differentiation of hematopoietic cells by sustaining GATA-2 expression. Blood 2001;98:3283–3289.[Abstract/Free Full Text]
- Cheng T, Shen H, Giokas D et al. Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells. Proceedings Of The National Academy of Sciences of the United States of America 1996;93:13158-13163.
- Hall MA, Slater NJ, Begley CG et al. Functional but abnormal adult erythropoiesis in the absence of the stem cell leukemia gene. Mol Cell Biol 2005;25:6355–6362.[Abstract/Free Full Text]
- Rodrigues NP, Janzen V, Forkert R et al. Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis. Blood 2005;106:477–484.[Abstract/Free Full Text]
- Qian F, Zhen F, Xu J et al. Distinct functions for different scl isoforms in zebrafish primitive and definitive hematopoiesis. Plos Biology 2007;5:e132.[CrossRef][Medline]
- Calkhoven CF, Muller C, Martin R et al. Translational control of SCL-isoform expression in hematopoietic lineage choice. Genes Dev 2003;17:959–964.[Abstract/Free Full Text]
Top
Abstract
Introduction
shRNA-Ctrl and