Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Abstract

Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function

“…As detailed in Figure 7, our real‐time PCR data is consistent with the notion that as OB culture duration increases so does OB maturation. Specifically we show that (1) Runx2 expression is highest in fresh OB cultures and decreases with increases in OB culture duration (Runx2 is a transcription factor which is critical for differentiation of early stage OB lineage cells) [Stein and Lian, 1993; Otto et al, 1997; Mundlos, 1999; Lian et al, 2004; Schroeder et al, 2005; Krause et al, 2008]; (2) alkaline phosphatase expression increases with OB culture duration up to 2 weeks (3 weeks cumulative OB culture duration) and then stabilizes; and (3) all of the OB‐specific bone matrix proteins tested showed an increase in expression with an increase in culture duration (type I collagen, osteopontin (OPN), and osteocalcin) suggesting that cells in the latter culture durations are mature OB producing osteoid while fresh OB are early stage OB lineage cells.…”

Novel method for the isolation and characterisation of the putative prostatic stem cell

“…Cell cycle characteristics of prostate SP tail cells were evaluated by plotting Hoechst 33342 (DNA content) along the x‐axis and Pyronin Y (RNA content) along the y‐axis (50). This method isolates cells in G 0 /G 1 phase by Hoechst 33342 distribution and subfractionates by RNA content into G 0 (RNA low) and G 1 (RNA high) as determined by Pyronin Y staining.…”

High mobility group protein HMGA1 inhibits retinoblastoma protein‐mediated cellular G0 arrest

“…The quiescent state in the G0 phase is well-characterized by low levels of general transcription activities. (44) Therefore, we isolated total RNAs and genomic DNAs at each cell cycle stage, and measured RNA / DNA ratios (Fig. 3b).…”