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Commitment of stem cells to hematopoietic lineages: a functional genome analysis

Summary

Background: Hematopoietic stem cells (HSC) represent the common origin of all lineages of the human immune system. While many transcription factors and cytokines guiding commitment to and differention along various hematopoietic lineages have been identified in recent years its is not understood, how these factors integrate to determine the destiny of HSC. To date, a genome-wide analysis of early cell fate decisions within the bone marrow is missing.

Experimental outline:Using the SAGE technique, genome-wide gene expression profiles of HSC and all known lymphoid and myeloid progenitor populations will be compared to identify so far unrecognized candidate genes of lineage commitment. Using SAGE, also novel differentially expressed transcripts can be identified. The role of such transcripts can be studied under cell culture conditions in a functional screen based on RNA interference (RNAi). Novel cDNA fragments, whose RNAi-based silencing affects the differentiation program of primary stem and progenitor cell cultures will be extended to full-length cDNAs by 5’RACE and subjected to further functional characterization.

Goals: During a five-years research period, we aim at a genome-wide analysis of human hematopoiesis. The analysis is meant to be comprehensive as it will cover all currently known lineages arising from HSCs. The origin of HSCs from pluripotent stem cells will be particularly emphasized. Starting from genome-wide gene expression profiles established by SAGE, a systematic search for novel or previously unrecognized regulator genes of normal and malignant development originating from HSCs will be carried out. Identification and characterization of novel candidate genes will address the following aspects:

• Analysis of genetic reprogramming during commitment to specific lineages
• Genome-wide identification of yet unrecognized target-genes of lineage determination
• Characterization of the differentiation potential of pluripotent vs lineage-restricted stem cells
• Molecular definition of a common lymphoid progenitor in human cord blood
• Genome-wide analysis of early normal and malignant lymphoid development
• Commitment of pluripotent stem cells to hematopoietic vs non-hematopoietic lineages

Introduction

Hematopoietic stem cells (HSC) can give rise to about ten distinct blood cell lineages and can maintain blood production in mice and humans throughout lifetime. Between growth and differentiation, HSC regulate the balance between self-renewal and commitment to various lineages. The mechanisms that govern stem cell fate decisions are tightly regulated even though there is evidence for a differentiation potential of HSC beyond hematopoietic lineages (Orlic et al., 2001; Tsai et al., 2002).
Despite extensive stem cell characterization, only very little is known about the molecular nature of these regulatory mechanisms. Individual molecules have been shown to be required for early aspects of hematopoietic development, but it has not been possible to delineate regulatory pathways that function at the level of self-renewing hematopoietic stem cells (Flake et al., 1986; Guenechea et al., 2001). In order to understand, how individual transcription factors and signaling molecules integrate to determine the destiny of HSC, a genome-wide description of the molecular components available to the stem cell is required, that is, its genetic program.

Transcriptional regulation of early hematopoietic development

Among primitive stem cells, the Lin^- population expressing both SCA1 and KIT (the so-called SKL cells) likely comprise most if not all cells with HSC potential. Among SKL cells, at least two hematopoietic stem cell (HSC) subsets can be distinguished based on differential expression of CD34 and CD38: While CD34⁺ CD38^low SKL cells provide immediate radioprotection for lethally irradiated mice, CD34^low CD38⁺ SKL cells are responsible for long-term reconstitution of both myeloid and lymphoid lineages.
Previous work based on mouse models identified a number of transcriptions factors that are essential for early hematopoiesis. While SCL (stem cell leukemia hematopoietic transcription factor; Gottgens et al., 2002), GATA2, LMO2 and FLK1 (Shalaby et al., 1997) are required for both primitive and definitive hematopoiesis, transcriptional activation through AML1 and its partner CBFb are specifically necessary for definitive hematopoiesis (Zhu and Emerson, 2002). While these transcription factors are indispensible to initiate early hematopoietic development, other molecules including HOXB4, IKAROS, NOTCH1 and its ligand JAGGED1, BMP4 (Bhatia et al., 1999) and TNFR1 strongly impact on the self-renewal capacity of HSC (Zhu and Emerson, 2002).

The origin of common lymphoid and myeloid lineages

Once HSC divide and generate more differentiated daughter cells, within 10 to 15 divisions the genetic programs of their progeny becomes determined towards a distinct lineage. How restriction of HSC "plasticity" to a unique lineage functions, is unkown as yet. However, it has been proposed that restriction to a common myeloid vs a common lymphoid differentiation pathway precedes final lineage commitment.
The fate decision between common myeloid (CMP) and common lymphoid progenitors (CLP) can be directed by signals through the IL7 receptor, which effectively suppresses myeloid differentiation. Indeed, IL7R⁺ Lin^- SCA1^low KIT^low CLP possess short-term repopulating ability restricted to B, T or NK cell precursors. Of note, GATA3 and IKAROS determine lymphoid development while GATA1, CEBPa, NFE2, FOS (Grigoriadis et al., 1994) and the GM-CSF receptor initiate and maintain myeloid differentiation. CMP (IL7R^- Lin^- SCA^- KIT⁺) can give rise to monocytoid, granulocytic and erythroid progenitors as well as megakaryocytes (see Figure 12).

Specific requirements for myeloid and lymphoid differentiation

CMP differentiate into either common precursors for monocytic and granulocytic lineages (GMP) or common precursors for both erythroid and megakaryocytic lineages (EMP). Concomitant expression of PU.1 and GATA1 leads HSC to CMP, while subsequent predominance of either PU.1 or GATA1/ FOG directs differentiation to GMP or EMP, respectively. Within the GMP-compartment, the distinction between monocytic and granulocytic development depends on the presence or absence of sustained expression of CEBPa, which determines granulocytic differentiation. Lymphoid commitment most importantly depends on IL-7-mediated downregulation of GM-CSF receptor expression. CLPs that are not further committed to either the B or T lineage typically express the common acute lymphoblastic leukemia antigen (CALLA) CD10 and lack expression of the earliest unambiguous B (CD19) or T (CD7) lineage markers.
NOTCH1 and DELTEX commit bone marrow CLP to the T lineage (Radtke et al., 1999). These T lineage precursors have not yet rearranged T cell receptor genes and are thought to leave the bone marrow to immigrate into the thymus, where commitment reaches an irreversible stage upon expression of a pre-T cell receptor after productive rearrangement of TCRb V, D and J segments. Until expression of a TCRb chain within the pre-TCR complex together with pre-Ta, T lineage commitment is leaky and coexpression of B lineage markers such as Iga and Igb on the cell surface is a common feature (Wang et al., 1998).
Determination of B cell differentiation critically requires consecutive upregulation of E2A, EBF and, most importantly, PAX5 (Nutt et al., 1999). While E2A and EBF are expressed only transiently during early B cell development within the bone marrow, the expression of PAX5 is required to finalize the commitment decision and to maintain lineage identity throughout the life of a B cell. While expression of the pre-T cell receptor marks the decisive step in the commitment process, definitive lineage commitment precedes rearrangement of immunoglobulin heavy chain (IgH) genes. Driven by PAX5, committed B cell progenitors already express the B cell antigen CD19 with both IgH alleles in germline configuration (Allman et al., 1999). Subsequently, pro-B cells exhibit an active recombination machinery with rearrangement of IgH D to J segments, while pre-B cells carry an IgH VDJ rearrangement encoding a functional heavy chain (Rolink et al., 1991).