G. Brady et al., ANALYSIS OF GENE-EXPRESSION IN A COMPLEX DIFFERENTIATION HIERARCHY BYGLOBAL AMPLIFICATION OF CDNA FROM SINGLE CELLS, Current biology, 5(8), 1995, pp. 909-922
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 po
pulations or cell lines which may also be heterogeneous, and the criti
cal differences in gene expression that might determine divergent deve
lopment are obscured. In this study, we combined global amplification
of mRNA transcripts in single cells with identification of the develop
mental 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 diff
erentiative potentials; sibling cells generally each formed clones of
identical composition in secondary culture. Globally amplified cDNA wa
s prepared from individual precursors whose developmental potential wa
s identified by tracking sibling fates. Further cDNA samples were prep
ared from terminally maturing, homogeneous hemopoietic cell population
s. Together, the samples represented 16 positions in the hemopoietic d
evelopmental hierarchy. Expression patterns in the sample set were det
ermined for 29 genes known to be involved in hemopoietic cell growth,
differentiation or function. The cDNAs from a bipotent erythroid/megak
aryocyte precursor and a bipotent neutrophil/macrophage precursor were
subtractively hybridized, yielding numerous differentially expressed
cDNA clones. Hybridization of such clones to the entire precursor samp
le set identified transcripts with consistent patterns of differential
expression in the precursor hierarchy. Conclusions: Tracking of sibli
ng fates reliably identifies the differentiative potential of a single
cell taken for PCR analysis, and demonstrates the existence of a vari
ety of distinct and stable states of differentiative commitment. Globa
l amplification of cDNA from single precursor cells, identified by sib
ling fates, yields a true representation of lineage- and stage-specifi
c gene expression, as confirmed by hybridization to a broad panel of p
robes. 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 int
roduce a powerful set of tools for unravelling the genetic basis of li
neage divergence.