Abstract
Hematopoietic stem cells (HSCs) are probably the best-studied adult tissue-restricted stem cells. Although methods for flow cytometric detection of phosphoproteins in hematopoeitic progenitors and mature cells are available, analogous protocols for HSC are lacking. We present a robust method to study intracellular signaling in immunophenotypically-defined murine HSC/progenitor cell (HPC)-enriched populations. Using this method, we uncover differences in the response dynamics of several phosphoproteins representative of the Ras/MAP-Kinase(K), PI3K, mTOR and Jak/STAT pathways in HSC/HPCs stimulated by Scf, Thpo, as well as several other important HSC/HPC agonists.
Highlights
Hematopoietic stem cells (HSCs) self-renew and give rise to all hematopoietic cells
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/HSC/progenitor cell (HPC) populations
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, multipotent progenitors (MPP), granulocyte/monocyte progenitors (GMPs)/common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs)), and intracellular parameters in HSC/HPC, akin to studies of more mature hematopoietic cells [6]
Summary
HSCs self-renew and give rise to all hematopoietic cells. Adult murine HSCs can be prospectively enriched by their low expression of multiple lineage markers (LIN2) including CD127, and high expression of c-Kit (hereafter, Kit) and Sca-1, as well as several other markers [1]. Can be further purified from short-term (ST)-HSC and multipotent progenitors (MPP) (CD34+) using CD34 antibodies. MPPs give rise to lineage-restricted progenitors with minimal self-renewal and more restricted differentiation capacities, including common lymphoid progenitors (CLPs; LIN2c-KitloSca-1loCD34+CD127+), lymphoidprimed MPPs (LMPPs; LIN2c-Kit+Sca-1+CD34+Flt3+CD127+/2), and common myeloid progenitors (CMPs) [1]. CMPs (LIN2cKit+Sca-12CD34+FccRII/IIIloCD1272) yield granulocyte/monocyte progenitors (GMPs: LIN2c-Kit+Sca-12CD34+FccRII/ III+CD1272) and megakaryocyte/erythroid progenitors (MEPs:LIN2c-Kit+Sca-12CD342FccRII/III2CD1272), which give rise to all red and white blood cells, including platelets. The LIN2c-Kit+Sca-1+ (LSK) fraction of the bone marrow (BM) is enriched for HSCs and MPPs, whereas the LIN2c-Kit+Sca-12 (LK)
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