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M. Xie et al.
nificantly reduced that of CD34+/–FLT3– KSL cells.32 It also has been reported that deletion of TPO or Mpl results in the increase of cycling of HSC and subsequent reduction of the pool of quiescent HSC in mice.32,33
TPO in synergy with SCF promoted rapid division of HSC in vitro (Figures 2 and 5). SCF + TPO transiently increased the level of ST reconstitution but this was sub- stantially decreased in LT reconstitution (Figures 3A, 4D, and Online Supplementary Table S8). In this study, SCF + TPO supported the differentiation more than self-renewal in HSC. However, HSC may behave differently, depend- ing on different culture conditions as recently reported.34 It has also been recently reported that a low concentration of SCF and TPO can maintain HSC quiescent HSC in vitro.35 TPO may have different roles in the regulation of HSC under different conditions.
About 30% of HSC1 expressed Csf3r, but either G-CSF alone or G-CSF + SCF did not induce their division at all (Figure 2A and data not shown). SCF + G-CSF did not increase LT reconstitution level more than did SCF alone (Figures 3A, 4B and C, and Online Supplementary Tables S6 and S7). Csf3r expression was upregulated in SCF + G-CSF culture (Figure 5). The percentage of SOCS3+ cells increased in Csf3r+ cells after SCF + G-CSF culture (Online Supplementary Figure S4A). SOCS3 may play a negative role in G-CSF signaling in LT-HSC as reported for G-CSF- driven granulopoiesis.36,37 As a result, most Csf3r+ LT- HSCs do not respond to G-CSF. Only approximately 10% of Csf3r+ cells responded to G-CSF and continuously divid- ed more than three times and differentiated (Figure 2A), presumably escaping from the negative regulation by SOCS3. SCF + G-CSF maintained the reconstitution potential of HSC2 in vitro (Figure 3B), supporting a previ- ous study demonstrating that SCF + G-CSF maintains B- lymphoid potential in culture.38 Taken together, these data suggested that c-Kit and G-CSFR signaling in ST Ly-bi HSC is regulated differently from that in LT My-bi HSC.
Previously, HSC were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE), their division was fol- lowed by CFSE intensity, and the function of CFSE- labeled HSC was examined through serial transplantation. This in vivo HSC division tracking study reported that G- CSF did not induce self-renewing division of HSC.6 Similarly, the other group, using H2B-GFP label retaining system, also reported that G-CSF did not stimulate LT- HSC.7 Supporting these studies, this study showed that G- CSF did not directly act on LT-My-bi HSC in vitro.
from those of G-CSF, and particular progenitors can receive signals from both G-CSF and GM-CSF.
Cxcl12 is also considered to be a niche factor synthe- sized by bone marrow stromal cells.9,41,42 Deletion of Cxcl12 from the BM, or its receptor, Cxcr4, from hematopoietic cells reduced HSC in the BM, indicating their roles in HSC retention.9,41-43 In agreement with this, successful mobilization of HSC and HPC from BM into the circulation has been achieved by the use of G-CSF or Cxcr4 antagonists through the disruption of the Cxcr4/Cxcl12 interaction.44,45 In this study, however, we detected little expression of Cxcr4 in HSC (Figure 1C) even after stimulation with SCF, SCF + G-CSF, and SCF + TPO for 7 days (Figure 5). At least some HSC may not express Cxcr4 and be mobilized via Cxcr4-independent mechanism. As Cxcr7 has been reported as a new candi- date receptor for Cxcl12.46 Nevertheless, more precise mechanisms of HSC mobilization by G-CSF should be clarified.
Hematopoiesis is a blood formation process depicted as a hierarchy with self-renewing HSC ranking at the apex. In our model (Figure 6), HSC1 cells resides at the top rank, HSC2 and HPC1 cells reside at the second rank, and HPC2, HPC3, and HPC4 cells reside at the third rank. SCF, G-CSF, GM-CSF, and TPO act on different cell types, sup- porting the concept that cytokines exhibit multiple func- tions affecting cells at different developmental stages.47 In addition, based on the cytokine responses, two major dif- ferentiation pathways from LT-HSCs are suggested: (1) the HSC1-HPC1-HPC2 pathway represents the myeloid differentiation pathway;19 and (2) the HSC1-HSC2- HPC3/4 pathway represents the lymphoid differentiation pathway. LT-HSC differentiated into either ST-HSC or myeloid progenitors like common myeloid progenitors. ST-HSC further give rise to LMPP which lose the response to SCF and TPO (Figure 2B), consistent with the decreased self-renewal ability and megakaryocyte potential in LMPP.48 G-CSF is involved in both the myeloid and lym- phoid differentiation pathways, regulating the prolifera- tion of myeloid progenitors while maintaining Ly-bi HSC. In this regard, G-CSF works as a multipotent factor in hematopoiesis. Collectively, this cytokine network model indicates that different cytokines play a role in different differentiation pathways.
This study showed that LT-My-bi HSC do not respond to G-CSF, suggesting that HSC in healthy donors are pro- tected from the proliferation and sequential exhaustion after G-CSF administration. G-CSF enhanced the efficacy of chemotherapy for eliminating leukemia stem cells without affecting the survival of normal HSC in the mouse acute myeloid leukemia (AML) model.49 A large randomized clinical trial of G-CSF in AML patients showed that priming AML cells with G-CSF reduced the rate of relapse and improved disease-free survival without affecting hematologic recovery.50 These studies together with our own suggested that normal HSC are protected from the cytotoxic effect of chemotherapy when G-CSF is injected before chemotherapy.
Disclosures
No conflicts of interest to disclose.
Contributions
MX performed the experiments, acquired and analyzed data, and wrote the manuscript; SZ helped with the transplantation
Our conclusions of the G-CSF effect may contradict pre- vious studies.3-5,10 The levels of previous HSC purifications were not as high as ours, as they contained a mix of LT- My-bi HSC, ST-Ly-bi HSC, and HPC; serum was used in culture; some experiments lacked an appropriate control; the follow-up period after transplantation was not long enough to distinguish ST-Ly-bi HSC from LT-My-bi HSC by our definition; and experimental conditions differed. These differences may account for the discrepancies.
GM-CSF receptor is composed of two subunits, α and b. The α subunit binds GM-CSF with low affinity, while b subunit has no binding capacity by itself but forms a high affinity receptor with α subunit and plays a role in signal transduction.39,40 Our data showed that most HSC and HPC populations did not express Csf2ra, but most HPC1 and half of HSC2, HPC3, and HPC4 expressed Csf2rb. Only HPC2, HPC3 and HPC4 responded to GM-CSF. These data showed that target cells of GM-CSF differ
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