Gerard Brady

Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells

BACKGROUND Many differentiating tissues contain progenitor cells that differ in their commitment states but cannot be readily distinguished or segregated. Molecular analysis is therefore restricted to mixed populations or cell lines which may also be heterogeneous, and the critical differences in gene expression that might determine divergent development are obscured. In this study, we combined global amplification of mRNA transcripts in single cells with identification of the developmental potential of processed cells on the basis of the fates of their sibling cells from clonal starts. RESULTS We analyzed clones of from four to eight hemopoietic precursor cells which had a variety of differentiative potentials; sibling cells generally each formed clones of identical composition in secondary culture. Globally amplified cDNA was prepared from individual precursors whose developmental potential was identified by tracking sibling fates. Further cDNA samples were prepared from terminally maturing, homogeneous hemopoietic cell populations. Together, the samples represented 16 positions in the hemopoietic developmental hierarchy. Expression patterns in the sample set were determined for 29 genes known to be involved in hemopoietic cell growth, differentiation or function. The cDNAs from a bipotent erythroid/megakaryocyte precursor and a bipotent neutrophil/macrophage precursor were subtractively hybridized, yielding numerous differentially expressed cDNA clones. Hybridization of such clones to the entire precursor sample set identified transcripts with consistent patterns of differential expression in the precursor hierarchy. CONCLUSIONS Tracking of sibling fates reliably identifies the differentiative potential of a single cell taken for PCR analysis, and demonstrates the existence of a variety of distinct and stable states of differentiative commitment. Global amplification of cDNA from single precursor cells, identified by sibling fates, yields a true representation of lineage- and stage-specific gene expression, as confirmed by hybridization to a broad panel of probes. The results provide the first expression mapping of these genes that distinguishes between progenitors in different commitment states, generate new insights and predictions relevant to mechanism, and introduce a powerful set of tools for unravelling the genetic basis of lineage divergence.

Routine expression profiling of microarray gene signatures in acute leukaemia by real-time PCR of human bone marrow

Cancer subtype diagnosis using microarray signatures has the potential to transform pathological diagnosis but the routine measurement of genes signatures remains difficult. Reverse transcription polymerase chain reaction (RT‐PCR) measurement of Indicator genes for acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) was used to determine gene signatures. Bone marrow (BM) mononuclear cells were sorted into total, CD34+ and CD34− fractions, and mRNAs globally amplified from each fraction using polyA PCR. The expression profile of the 17 top‐ranked genes distinguishing AML and ALL were measured by RT‐PCR in five ALL, 26 AML, 12 AML remission, four chronic myeloid leukaemia (CML) and nine morphologically normal BM samples. All but two of the genes measured showed similar expression in AML and ALL to that reported previously. Specifically, c‐MYB (P ≤ 0·04) was significantly increased in ALL in the total fraction, whilst HOXA9 (P ≤ 0·19) and cystatin c (P ≤ 0·01) were increased in AML in the CD34+ and CD34− fractions, respectively. c‐MYB, hSNF2, RBAP48, HKRT‐1, LYN, CD33, Adipsin and HOXA9 were increased in AML compared with remission AML, indicating an ability to determine disease activity. The method used is simple, sensitive and robust, enabling routine clinical use, and it can also be extended to other tumours types with gene signatures.

The LSP1 gene is expressed in cultured normal and transformed mouse macrophages

The mouse LSP1 protein is an F-actin binding protein initially isolated as a cDNA from the BALB/c pre-B cell line 220.2. Its expression pattern is highly restricted. Only lymphocytes and lymphoma cell lines were reported to express LSP1. Non-lymphoid cell lines or normal mouse tissues such as brain, lung, liver, skeletal muscle, kidney or testis do not express LSP1. Here we report that mouse macrophage cell lines also express LSP1 mRNA and protein. DNA sequence analysis shows that the coding sequence of LSP1 RNA expressed in the macrophage cell line P388D1 is identical to the sequence of LSP1 RNA from the pre-B cell line 220.2. To determine the expression of LSP1 RNA in normal macrophages derived from fetal liver and adult bone marrow and in other hematopoietic cells we used a recently described technique to make representative amplified polyA cDNA samples from small numbers of cells or isolated hematopoietic colonies. Analysis of these cDNA samples with an LSP1 cDNA probe showed that eight of nine macrophage samples expressed LSP1 RNA. One of two neutrophil samples but none of eight other non-lymphoid colonies was positive for LSP1 RNA. From these results it appears that the expression of LSP1 RNA in the hematopoietic system is restricted to the lymphocyte, macrophage and neutrophil lineages.

Expression of basic helix-loop-helix transcription factors in explant hematopoietic progenitors.

The basic helix‐loop‐helix (bHLH) transcription factors form heterodimers and control steps in cellular differentiation. We have studied four bHLH transcription factors, SCL, lyl‐1, E12/E47, and Id‐1, in individual lineage‐defined progenitors and hematopoietic growth factor—dependent cell lines, evaluating mRNA expression and the effects of growth factors and cell cycle phase on this expression. Single lineage‐defined progenitors selected from early murine colony starts and grown under permissive conditions were analyzed by RT‐PCR. SCL and E12/E47 were expressed in the vast majority of tri‐, bi‐, and unilineage progenitors of erythroid, macrophage, megakaryocyte, and neutrophil lineages. Expression for E12/E47 was not seen in unilineage megakaryocyte and erythroid or bilineage neutrophil/mast cell progenitors. Lyl‐1 showed a more restricted pattern of expression, although expression was seen in some bi‐ and unilineage progenitors. No expression was detected in erythroid, erythroid‐megakaryocyte‐macrophage, macrophage‐neutrophil, macrophage, or megakaryocytic progenitors. Id‐1, an inhibitory bHLH transcription factor, was also widely expressed in all bi‐ and unilineage progenitors; only the trilineage erythroid‐megakaryocyte‐macrophage progenitors failed to show expression. Expression of these factors within a progenitor class was generally heterogeneous, with some progenitors showing expression and some not. This was seen even when two sister cells from the same colony start were analyzed. Id‐1, but not E12/E47, mRNA was increased in FDC‐P1 and MO7E hematopoietic cell lines after exposure to IL‐3 or GM‐CSF, Id‐1, E12, and lyl‐1 showed marked variation at different points in cell cycle in isoleucine‐synchronized FDC‐P1 cells. These results suggest that SCL, lyl‐1, E12/E47, and Id‐1 are important in hematopoietic progenitor cell regulation, and that their expression in hematopoietic cells varies in response to cytokines and/or during transit through cell cycle.

Gene expression analysis of myeloid and lymphoid lineage markers during mouse haematopoiesis

Expression profiling of haematopoietic cells is hampered by the heterogeneous nature of haematopoietic tissues and the absolute rarity of early unrestricted progenitors. To overcome this, the expression profile of lymphoid and myeloid‐associated genes (LEF1, EBF, CD19, Sox‐4, B29, CD45, C‐fms, lysozyme, PU.1 and CD5) were investigated in 40 mouse myeloid haematopoietic precursors covering the entire haematopoietic hierarchy from multipotential to committed single lineages. The lineage‐specific expression seen in single‐cell studies was confirmed by examining fractionated bone marrow, whole tissues and differentiation of the multipotent cell line FDCP (Factor Dependent Cell Paterson) mix. Analysis of the 40 single myeloid precursors failed to detect expression of lymphoid‐associated genes, LEF1, EBF, CD19 and CD5, despite detection in lymphoid cell controls. Surprisingly, the lymphoid‐associated genes, Sox‐4 and B29 were detected in the single myeloid precursors, which was confirmed in bone marrow and a multipotential myeloid cell line. The pattern of Sox‐4 and B29, is consistent with a potential role in the commitment of bipotential granulocytic/macrophage precursors towards the granulocyte or macrophage lineage. In addition to providing baseline values for myeloid and lymphoid lineage markers during mouse haematopoiesis, these results highlight the importance of single‐cell analysis in the study of complex tissues.