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Purpose: The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB)-derived progenitors, used in transplantation. Methods: We correlated gene expression patterns of highly enriched steady-state peripheral blood (PB)- and mPB-derived CD71(+) cells by microarray and ingenuity pathway analyses, to identify the transcriptional program during in vitro erythroid differentiation. Results: The gene expression was more than doubled in mPB-derived (4180 genes) compared to PB-derived erythroid progenitors (1667 genes) while PB-and mPB-derived erythroid progenitors shared 1534 common genes. Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin, where we identified common 13 downregulated and 30 upregulated genes. The most significant genes in mPB-derived erythroid progenitors were P4HB, DDIA3, ARPC2 and ATP5G3. Regarding G-CSF stimulation the G-CSF receptor CSF2RB (1.1-fold) was linked via STAT3 to erythroid-specific ALAS2 (2.9-fold) and GATA2 (1.3-fold) factors, all upregulated in mPB-derived erythroid progenitors, coupled to common upregulated NUDC gene involved in the proliferation of erythroid cells. Conclusion: This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources.

Different biological functions of hematopoietic cells are regulated by signals cells receive from their environment that may come from interactions with other cells or from soluble growth factors and cytokines. Although the effects of hematopoietic regulators are well described, the exact intracellular signaling cascades leading to various cellular responses are not fully elucidated. All hematopoietic growth factors and cytokines seem able to activate all the major signal transduction pathways simultaneously, and it appears that the same network of signaling proteins may coordinate numerous cellular functions. However, the differences that lead to the unique biological events of the particular cytokine, as well as the differences that determine lineage-specific blood cell differentiation are only gradually being uncovered. With the help of specific inhibitors of known signal transduction pathways, we have examined the contribution of particular signaling molecules of Jak/Stat, MAP kinase and PI-3 kinase pathways, as well as the activation of nuclear factor kappaB (NFkB) transcription factor on the proliferation and differentiation of murine bone marrow granulocyte-macrophage (CFU- GM) and erythroid (BFU-E and CFU-E) progenitors. Preliminary results demonstrated that the effects of the inhibitors on the hematopoietic colony formation were lineage-dependent, as well as dependent on erythroid progenitors' stage of differentiation. The obtained differences suggest that different signal transduction intermediates regulate erythroid and myeloid progenitor cell proliferation and differentiation. Differences in the susceptibility of the progenitor cells to the inhibitors used were also observed. The data is consistent with other evidences indicating that different threshold levels are one of the mechanisms by which the signaling specificity may be achieved. Mnogobrojne funkcije hematopoetskih ćelija u toku procesa hematopoeze, regulisane su brojnim signalima koje ćelije primaju iz svoje okoline i to kako od drugih ćelija tako i od brojnih regulatornih molekula. Iako su biološki efekti regulatora hematopoeze uglavnom dobro opisani, putevi prenošenja signala unutar ćelija na koje deluju nisu još uvek u potpunosti razjašnjeni. Savremena istraživanja ukazuju da gotovo svi poznati hematopoetski regulatori mogu da aktiviraju gotovo sve bitne puteve prenošenja signala unutar ćelija, što sve upućuje da ista mreža signalnih komponenti može da koordinira brojne ćelijske funkcije. Međutim, razlike u transdukciji signala koje dovode do jedinstvenog biološkog dejstva svakog citokina, kao i razlike u prenosu signala kojom se određuje lozno-zavisna diferencijacija krvnih ćelija nisu još poznati. Naša istraživanja započela su određivanjem učešća pojedinih komponenti JAK/STAT puta signalizacije, MAP kinazne kaskade i PI-3 kinaznog puta, kao i aktivacije transkripcionog faktora NFKB, u proliferaciji i diferentovanju opredeljenih matičnih ćelija granulocitno-monocitne (CFU-GM) i eritrocitne (BFU- E i CFU-E) loze kostne srži normalnih miševa. Početni rezultati ovih ispitivanja su ukazali na postojanje kako lozno-zavisnih razlika u odgovoru matičnih ćelija hematopoeze na pojedine inhibitore, tako i na zavisnost odgovora od stepena zrelosti matičnih ćelija u okviru iste loze. Dobijene razlike upućuju da su različiti učesnici signalnih puteva uključeni u regulaciju proliferacije i diferencijacije eritrocitnih i granulocitno-monocitnih progenitora. Ovo je jedan od mogućih načina kojim se možda omogućava selektivna ekspanzija ćelija određenih krvnih loza u zavisnosti od potreba organizma. Takođe, uočene su i razlike u pragu osetljivosti različitih kategorija opredeljenih matičnih ćelija hematopoeze na ispitivane inhibitore signalnih puteva, što ide u prilog podacima da su različiti pragovi osetljivosti jedan od mogućih mehanizama kojim se najverovatnije ostvaruje specifičnost delovanja signalnih puteva.

Steady-state hematopoiesis takes place in the bone marrow permissive microenvironment, composed of stromal cells, as well as extracellular matrix (ECM) components and regulatory molecules, produced by both stromal and hematopoietic cells. Stromal cells are both mezenchymal (endothelial cells fibroblasts, adipocytes, osteoblasts, myocytes, adventitial reticular cells) and hematopoietic (macrophages) in origin, which together provide cell to cell interactions, matrix proteins and growth factors essential for the maintenance, growth and differentiation of hematopoietic stem and progenitor cells. The ECM components (collagen, laminin, fibronectin, thrombospondin proteoglycans, hemonectin and various proteinases participating in their remodeling) are involved in different biological functions such as cell adhesion, binding and presentation of various cytokines and regulation of cell growth. These components serve to localize hematopoietic cells in the specific bone marrow microenvironment and it is suggested that in combination with cytokines are crucial for their compartmentalization. The regulatory molecules of hematopoietic microenvironment are cytokines, which regulate the survival, proliferation and differentiation of hematopoietic cells and cell adhesion molecules, which are responsible for the localization of hematopoiesis to the bone marrow. The activity of cytokines may be greater when the cytokine is present in a membrane-bound or ECM-associated form. Hematopoietic cells could also regulate normal hematopoiesis in an autocrine/paracrine manner, since it was shown that these cells express and secrete various growth factors, cytokines and chemokines. Proces hematopoeze se odvija u definisanoj tkivnoj mikrosredini kostne srži koja ima ključnu ulogu u njegovoj regulaciji. Mikrosredina hematopoeze podrazumeva funkcionalno jedinstvo stromalnih ćelija i ćelijskih produkata (molekuli ekstracelularnog matriksa i regulatorni faktori), koji čine kompleksni molekularni milje u kome se ostvaruju specifične interakcije hematopoetskih ćelija i komponenti mikrosredine. Stromalne ćelije mezenhimalnog porekla (endotelne ćelije, fibroblasti, adipociti osteoblasti, miociti, adventicijske ćelije retikuluma) i nemezenhimalnog porekla (makrofage), deluju na matične ćelije hematopoeze direktno među ćelijskim interakcijama, kao i produkcijom i deponovanjem pojedinih komponenti kompleksnog ekstracelularnog matriksa, a takođe i produkcijom i koncentrovanjem lokalnih citokina i faktora rasta sa hematopoetskim efektima, obezbeđujući na taj način gotovo sve činioce neophodne za proliferaciju i diferencijaciju matičnih ćelija hematopoeze. Komponente ECM (kolagen, laminin, fibronektin, trombospondin, proteoglikani, hemonektin i drugi glikoproteini kao i proteolitički enzimi koji omogućavaju remodelovanje ECM) su uključene u ćelijsku adheziju, vezivanje i prezentaciju različitih citokina i regulaciju ćelijskog rasta. Smatra se da komponente ECM omogućavaju lokalizuju hematopoetskih ćelija u specifičnoj mikrosredini kostne srži, i da u kombinaciji sa citokinima imaju presudnu ulogu u formiranju specifičnih niša. Regulatorni faktori hematopoetske mikrosredine su citokini koji regulišu preživljavanje, proliferaciju i diferencijaciju hematopoetskih ćelija i ćelijski adhezivni molekuli koji su odgovorni za lokalizaciju hematopoeze u kostnoj srži i za posredovanje u fizičkoj vezi između hematopoetskih ćelija i stromalnog tkiva mikrosredine. Ovi molekuli se u mikrosredini kostne srži nalaze kao solubilni, ili vezani za membrane stromalnih ćelija ili za komponente ECM, što može da pojača njihovu aktivnost. Takođe i same hematopoetske ćelije učestviju u regulaciji procesa hematopoeze produkujući citokine koji ispoljavaju autokrino/parakrine efekte.