We have developed a protocol that utilizes the multi-parametric and quantitative attributes of FACS to examine multiple phosphorylation events at the single cell level in highly purified HSC/HPC populations. Previous studies have only been able to analyze signaling events in bulk hematopoietic progenitors by flow cytometry 
or one cellular parameter at a time by immunofluorescence-based techniques 
. Although still limited by low target cell numbers and loss of a significant proportion of the starting material post fixation/permeabilization and washing steps, our protocol permits the simultaneous analysis of multiple cellular (HSC, MPP, GMP/CMP, MEP), and intracellular parameters in HSC/HPC, akin to studies of more mature hematopoietic cells 
Intracellular signaling networks in HSC/HPC have remained largely unexplored territory, mostly due to the rarity of these cell populations. Here we have analyzed signal transduction responses to several HSC/HPC agonists, including Scf and Thpo, both of which are important in vivo
for HSC function and can promote survival and enhance the proliferation of HSC/HPC ex vivo 
. Although Scf elicited rapid responses in some intracellular pathways that we interrogated (Ras/MAPK, PI3K/AKT) in all of the HSC/HPC cellular subsets we analyzed, Thpo stimulation profiles were stronger in HSC/MPP (compared to GMP/CMP or MEP) and consisted of the activation of a broader array of intracellular pathways (Ras/MAPK, PI3K/AKT, JAK/STAT). Other agonists, such as Flt3L, appeared to elicit stronger responses in LSK compared to LK cells, whereas Gm-csf elicited stronger responses in LK cells. These differences in response could reflect a heterogeneous expression of receptors in LSK and LK cells, as well as differences in the intrinsic sensitivity to these agonists between populations. We detected a significant synergistic effect of Scf and Thpo relative to either treatment alone on pS6 levels in LSK, resulting in a sustained pS6 response (). It is tempting to speculate that the reason for biological synergy (in terms of colony formation or proliferation/survival of HSC/HPC) previously observed between Scf and Thpo 
reflects sustained Ras/MAPK and mTOR signaling, although further genetic- or pharmacology-based biological assays are required to explore this point. Recent studies have demonstrated that both Thpo and Kit are required to maintain the steady-state pool of adult quiescent HSC 
. While STAT5 plays a significant role in HSC function 
, the role of members of the Ras/MAPK or mTOR pathway in HSC biology remains largely unexplored. Our data suggest that these pathways likely play a role downstream of agonist stimulation in HSC/HPC.
We also simultaneously assessed responses in pS6/pSTAT5 in CD34-LSK and LK subsets (). In all populations analyzed, approximately half of the cells respond to Thpo stimulation by elevating both pS6 and pSTAT5, but the rest only elevate pS6 (). This could reflect the relative hypersensitivity of the pathways downstream of Thpo leading to S6 phosphorylation over signaling to STAT5, lower sensitivity of our conditions for detecting pSTAT5, or intrinsic heterogeneity in the cell types present in these compartments. Regardless, the physiological relevance of this heterogeneity in signaling responses merits further investigation.
Our approach can be used along with selective inhibitors to examine pathway-dependencies. For example, our analysis of Scf-evoked S6 phosphorylation in primary HSC/HPC is largely in agreement with that of Roux et al. 
, who demonstrated that serum-evoked S6 phosphorylation at Ser235/236 in human cell lines (HEK 293) is dependent on an early MEK-dependent pathway (through RSK) and a later MEK- and mTOR-dependent pathway. Our data are also consistent with the Mek-inhibitor sensitivity of Ser235/236 phospohorylation in mouse embryonic fibroblasts doubly deficient for the mTOR downstream kinases S6K1/2 
. Our results suggest a model in which Scf stimulation leads to ERK activation that likely leads to RSK1/2 activation and S6 phosphorylation at Ser235/236 at 5 minutes post stimulation. At 15 minutes, S6 phosphorylation, is still partially UO126- and rapamycin- sensitive, although levels of pERK have returned to baseline at this time point. ERK activation also can lead to TSC2 phosphorylation and inactivation 
. Our data suggests the intriguing possibility that ERK mediated TSC2 inactivation is required along with mTOR-dependent phosphorylation of S6 to sustain high levels of pS6 in HSC/HPC. Direct testing of this model will be possible with the availability of appropriate phosphospecific antibodies.
Finally, our protocol should be amenable to the study of signal transduction abnormalities in leukemia stem cells (LSC), as these cells often share phenotypic similarities with normal HSC/HPC 
. The effects of drugs that may affect deregulated signal transduction pathways in leukemia can be readily tested in cell populations expressing surface epitopes examined in this study.