一、The bone marrow niche for haematopoietic stem cells
1、（P4） These studies suggested that endothelial cells are one component of the HSC niche, but did not address whether they directly or indirectly regulate HSC maintenance in vivo.
2、（P5）These results proved there is a perivascular niche for HSCs in which endothelial cells and mesenchymal cells promote HSC maintenance by synthesizing SCF.
3、（P5）These data confirmed that HSCs reside in a perivascular niche in which mesenchymal stromal cells and endothelial cells each synthesize multiple factors that promote HSC maintenance and localization.
4、（P5）The main sites of thrombopoietin synthesis are in the liver and kidney, although it is also synthesized at lower levels by bone marrow stroma104,105
5、（P6） Deletion of Gsα in osteoblastic cells, which is necessary for parathyroid hormone receptor signalling, markedly depleted pro- and pre-B cells in a way that could be mitigated with IL-7 (ref. 111）
6、（P6）Another set of questions concerns the manner in which the niche participates in diseases of stem-cell failure, such as aplastic anaemia or neoplasia
7、（P3Figure3） HSCs are found mainly adjacent to sinusoids throughout the bone marrow27,30,31,33, where endothelial cells and mesenchymal stromal cells promote HSC maintenance by producing SCF64, CXCL12 (refs 17, 33, 62) and probably other factors.
二、Complexity of bone marrow hematopoietic stem cell niche
1、(P1)Not only for physiologic state, we also discuss the recent evidences suggesting the importance of microenvironment for emergence of malignant hematopoietic diseases.
3、（P6）In the case of inducible BCR-ABL transgenic CML mice, direct contact of leukemic cells with MSCs was shown to allow the latter cells to expand their osteoblastic lineage cells (OBCs), characterized by alteration of several cytokine signaling pathways, including chemokine ligand 3 (CCL3) and TPO overproduction [53]. These remodeled OBCs have compromised capacity to support HSCs, while LSCs are maintained in such environment. These pieces of evidence have made it clear that LSCs not only occupy the HSC niche, but also remodel it into a self-reinforcing malignant microenvironment at the expense of normal hematopoiesis (Fig. 3a).
4.(P8)Together with an evidence that stromal cell populations isolated from individuals with myeloid malignancies can harbor genetic abnormalities that are different from mutations in leukemic clones [58], LSC niche should be added to the target of treatment for hematological malignancies.
5.(P8)At least two strategies can be envisioned to implement such notion: (1) detachment of LSCs from their niche and (2) targeting MSC remodeling and/or inflammation in the microenvironment. The efficacy of CXCR4 inhibitor AMD3100 (plerixafor) was already tested in AML patients in a non-randomized phase I/II study, which shows that the rate of overall complete remission or complete remission with incomplete blood count recovery was 46% in patients treated with the combination of this drug and the standard chemotherapy, indicating that this approach is feasible in AML [59]. Regarding the second strategy, there are currently no FDA-approved drugs that are able to directly target MSC remodeling. Considering that several cytokines including tumor necrosis factor α, IL-1, and IL-6 are involved in niche remodeling [53], it will be exciting to test the effect of blocking these cytokines with the use of antibodies that are available in the clinics.
三、Myeloproliferative Neoplasia Remodels the Endosteal Bone Marrow Niche into a Self-Reinforcing Leukemic Niche
1、（P1）Multipotent stromal cells (MSCs) and their osteoblastic lineage cell (OBC) derivatives are part of the bone marrow (BM) niche and contribute to hematopoietic stem cell (HSC) maintenance. Here, we show that myeloproliferative neoplasia (MPN) progressively remodels the endosteal BM niche into a self-reinforcing leukemic niche that impairs normal hematopoiesis
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2、(P1)MPN expanded OBCs, in turn, exhibit decreased expression of many HSC retention factors and severely compromised ability to maintain normal HSCs, but effectively support LSCs.
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3、（P1）At steady state, HSCs primarily reside in the bone marrow (BM) cavity, where they interact with different types of stromal cells expressing important regulatory molecules including stem cell factor (SCF), C-X-C motif chemokine 12 (CXCL12, also called SDF1a), and transforming growth factor b (TGF-b)
4、（P1）Both Runx2-expressing OBCs (Chitteti et al., 2010) and perivascular MSC-like cells expressing either nestin (Nes)(Me´ ndez-Ferrer et al., 2010), high levels of CXCL12 (so-called Cxcl12-abundant reticular cells, or CAR cells) (Omatsu et al., 2010), or leptin receptor (Lepr)(Ding et al., 2012) have been shown to be important for HSC maintenance.
5、（P1）Deregulation of HSC activity is an important step in the development of myeloid malignancies (Passegue´ et al., 2003). This is particularly the case for myeloproliferative neoplasms (MPNs), a class of clonal disorders that are propagated by leukemic stem cells (LSCs) arising from transformed HSCs and carrying oncogenic lesions such as BCR/ABL for chronic myelogenous leukemia (CML) or activating Jak2 mutations for polycythemia vera (PV), essential thrombocythosis, and primary myelofibrosis (PMF)
6、（P1）In addition, changes in the signaling activity of OBCs can alter HSC numbers and cause lineage-specific defects in blood production
7、（P1）In particular, decreased expression of Cxcl12 by BM stromal cells is observed in chronic-phase CML
8、（P2）However, much remains to be understood about how leukemic hematopoiesis impacts the BM microenvironment and, in turn, how changes in the activity of specific BM niche cells contribute to MPN pathogenesis.Here, we use an inducible Scl-tTA::TREBCR/ABL double-transgenic mouse model of human chronicphase CML (Reynaud et al., 2011) to investigate the effect of MPN development on the endosteal BM niche.
四、Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches
1、（P1）Deletion of Cxcl12 from endothelial cells depleted HSCs but not myeloerythroid or lymphoid progenitors. Deletion of Cxcl12from perivascular stromal cells depleted HSCs and certain restricted progenitors and mobilized these cells into circulation. Deletion of Cxcl12 from osteoblasts depleted certain early lymphoid progenitors but not HSCs or myeloerythroid progenitors, and did not mobilize these cells into circulation.
内皮细胞中Cxcl12的缺失使造血干细胞减少，但不包括髓样或淋巴样祖细胞。血管周围基质细胞中cxcl12的缺失使造血干细胞减少，某些限制性祖细胞被激活进入循环。成骨细胞中Cxcl12的缺失使某些早期淋巴祖细胞衰竭，但没有使造血干细胞或髓样祖细胞衰竭，也没有使这些细胞进入循环。
2、（P1）HSCs occupy a perivascular niche and early lymphoid progenitors occupy an endosteal niche.
3、（P1）Combined deletion of Scf from both endothelial cells and perivascular stromal cells caused severe HSC depletion and anaemia. In contrast, conditional deletion of Scf from osteoblasts or haematopoietic cells did not affect HSC frequency or function. This proves that there is a perivascular niche for HSC maintenance and raises the question ofwhether other haematopoietic progenitors reside in distinct niches.
HSC没有SCF的支持后还可以生长，证明除了干细胞生长因子外，还有perivascular niche维持HSC的生长
4、（P1）Some have proposed that the physiological source ofCXCL12 for HSC maintenance is osteoblasts，whereas others have proposed that the source is perivascular stromal cells;however,
Cxcl12 has not yet been conditionally deleted fromany candidate niche cell. Thus, the cellular sources ofCXCL12 for the maintenance ofHSCs and lymphoid progenitors remain uncertain.
CXCL12的细胞来源目前尚不清楚
5、（P1）Cxcl12–DsRed expression overlappedwithendothelialmarker staining, suggesting that endothelial cells were one source ofCXCL12 (Fig. 1a–c); however, perivascular stromal cells also seemed to produce CXCL12 (Fig. 1a–c).
6、（P1）In Scfgfp/1;Cxcl12DsRed/1 mice, we found a strong overlap in Cxcl12–DsRed and Scf–GFP expression by perivascular cells throughout the bone marrow (Fig. 1d–f). From flow cytometry, virtually all Scf–GFP1 stromal cells were positive for Cxcl12–DsRed (Fig. 1j) and virtually all Cxcl12–DsRed1 stromal cells were positive for Scf–GFP (Fig. 1k). Perivascular HSC niche cells therefore produce both SCF and CXCL12.
7、（P1）Approximately 90% of CD45/TER1192 PDGFRa1 perivascular stromal cells expressed Cxcl12–DsRed(Fig. 1l). From flow cytometry, approximately 70% of VE-cadherin1 endothelial cells and 0.5% of CD45/TER1191 haematopoietic cells in the bone marrow also expressed Cxcl12–DsRed (Fig. 1m, n and Supplementary Fig. 1g).
8、（P1）EYFP1 perivascular stromal cells from Lepr–cre;loxP– EYFP mice expressed Cxcl12 at ,15,000-fold the level observed in unfractionated bone marrow(Fig. 1p). VE-cadherin1endothelial cells, Col2.3–GFP1 osteoblasts, and Cxcl12–DsRed1 haematopoietic cells expressed significantly lower levels of Cxcl12 at ,120-fold, ,13-fold and ,3-fold the levels observed in bone marrow cells
9、（P2）Cxcl12fl/fl mice significantly reduced white blood cell counts (Supplementary Fig. 4a), lymphocyte frequencies (Supplementary Fig. 4b), bone marrow cellularity (Supplementary Fig. 4c) and CD1501CD482LIN2SCA11c-KIT1 HSC4 frequency.
10、（P3）Cxcl12 expression by haematopoietic cells is therefore not required for the maintenance or retention of HSCs or restricted progenitors in adult bone marrow
HSC自身CXCL12的表达对HSC或成年人骨髓中的限制性祖细胞的维护或保留没有需要
11、（P3）Nestin–cre-expressing cells are therefore not a physiologically important source of CXCL12 for the maintenance of HSCs or restricted progenitors in the bone marrow. Although HSC niche cells do not express endogenous nestin, nestin–cre or nestin–creER, they do seem to express the nestin–GFP transgene, consistent with previously published results.
12、（P3）This probably reflects a requirement for CXCL12 produced by postnatal bone marrow endothelial cells because Cxcl12-deficient mice do not showHSC depletion during fetal development, only in postnatal bone marrow17,22. HSCs from Tie2–cre;Cxcl12fl/fl mice had normal frequencies of BrdU1 cells and Annexin V1 cells, suggesting that HSCs may be depleted in these mice through premature differentiation
产后骨髓内皮细胞CXCL12缺乏影响了HSC，HSC在缺乏CXCL12的时候增殖正常，提示HSC损耗可能是因为HSC过早分化
13、（P3）
移植了缺乏CXCL12的骨髓细胞后B系祖细胞的变化
14、（P4）Both functional genetic data and localization data therefore suggest that there is an endosteal niche for a subset of early lymphoid progenitors
15、（P5）Our data indicate that HSCs depend on a perivascular niche created
by endothelial cells and Lepr–cre-or Prx1–cre-expressing perivascular stromal cells, whereas some early lymphoid progenitors depend on an endosteal niche created by osteoblasts, and committed B-lineage progenitors depend on a distinct perivascular niche created by Prx1– cre-expressing stromal cells but not endothelial cells.
五、Advances in understanding the acute lymphoblastic leukemia bone marrow microenvironment: From biology to therapeutic targeting.
1、（P1）Nevertheless, these niches provide sanctuaries where subsets of leukemic cells escape chemotherapy-induced death and acquire a drug-resistant phenotype
2、（P1）We shall also highlight the emerging role of exosomes/microvesicles as efficient messengers for cell-to-cell communication in leukemia settings. Studies on the interactions between the BM microenvironment and ALL cells have led to the discovery ofpotential therapeutic targets which include cytokines/chemokines and their receptors, adhesion molecules, signal transduction pathways, and hypoxia-related proteins.
3、（P1）A better understanding ofthe contribution ofthe BMmicroenvironment to theprocess ofleukemogenesis and leukemia persistence after initial remission, may provide new targets that will allow destruction of leukemia cells without adversely affecting healthy HSCs
4、（P1）This is at least partly due to the fact that 25–30% of B-ALL adult patients display the BCR-ABL1 fusion protein, which portends a poorer prognosis, while only 2–10% of pediatric B-ALL patients display such protein
5、（P2）while the vascular niche, localized at the sinusoidal walls, is composed of C-X-C motif chemokine (CXCL) 12-abundant reticular cells (CAR), endothelial cells, nestin-positive (NES+) mesenchymal stromal cells (MSCs)
6、（P2）A better understanding of the contribution of microenvironmental signals to the process of leukemogenesis and leukemia relapse/persistence after initial treatment, may provide new therapeutic targets that should allow eradication of LICs without adversely affecting HSCs.
7、（P3）Osteoblastic niche is mainly involved in maintaining hematopoietic stem cell (HSC) quiescence, while vascular niche presumably promotes proliferation, differentiation, andmobilization ofHSCs.
Endothelial cells express Notch ligands and stem cell factor (SCF), the ligand of c-Kit, and sustain HSC self-renewal and proliferation.
CXCL12/C-X-C Receptor 4 (CXCR4) signaling is essential for both HSC homing and maintenance. CXCL12 is secreted by several stromal cell types, especially CAR cells (CXCL12-abundant reticular cells).
8、（P4）Endothelial cells also modulated HSC homeostasis and differentiation by producing a specific set of angiocrine factors. In particular, endothelial cells in which the serine/threonine protein kinase Akt was activated, supported HSC self-renewal and expansion by up-regulating fibroblast factor 2 (FGF2), insulin-like growth factor binding protein 2 (IGFBP2), Ang-1, BMP4, and desert hedgehog (DHH). In contrast, endothelial cells where the mitogen-activated protein kinase/extracellular signalregulated kinase (MEK/ERK) signaling cascade was up-regulated, stimulated the expression off actors that promoted HSCs differentiation, including Ang-2 and interleukin (IL) -6
9、（P4）CXCL12 is mainly produced by CAR cells, which are located both around endothelial cells and at the endosteum
10、（P4）HSC maintenance genes, including Cxcl12, Ang-1, IL-7, and osteopontin
11、（P4）CTGF-depleted MSCs expressed higher leptin (an adipocyte-derived hormone) and CXCL12 levels, as compared with their normal counterpart
12、（P4）These findings shed further light on the mechanisms that protect ALL cells in BM microenvironment, and suggest novel potential therapeutic approaches/strategies (e.g. targeting leptin-producing adipocytes, or blocking leukemia cell migration toward adipocytes with CXCR4 antagonists) for ameliorating current therapies.
13、（P5）CXCL12 was first cloned from a BM–derived stromal cell line and identified as a pre-B cell growth stimulating factor
14、（P5）The CXCL12/CXCR4 axis is involved in development and maintenance of healthy tissues and organs [85,86], however it appears to play an important role in the disseminationofboth solid tumors and hematopoietic disorders, including ALL .
In the BM microenvironment, CXCL12 is synthesized and released by osteoblasts, CAR cells, and NES+ MSCs
Finally, bothMEK/ERK andp38 MAPK kinase promoted T-ALLmigration through interactions with α2β1 integrin
15、（P5）Nevertheless, CXCL12 was able to cause a transient activation of p38 MAPK in a model of B-ALL.
In particular, a very high CXCR4 expression was detected in mature B-ALL
16、（P6）T140 and AMD3100 (Plerixafor) are two CXCR4 antagonists that were able to block CXCL12-driven chemotaxis both in the B-ALL cell line, NALM6, and in primary B-ALL cells
High levels of FLT-3 may be associated with worse prognosis in acute leukemias
These findings indicated that interruption of leukemia/BM-MSC signaling is critical for the treatment of MLL-R ALL
It has also been reported that B-ALL cell lines up-regulated surface expression of CXCR4 in response to chemotherapeutic drugs (Ara-C, daunorubicin, vincristine).
CXCR7 (the second CXCL12 receptor, but also the receptor for CXCL11) in B-ALL cell lines
CXCR4 is highly expressed in human T-ALL cell lines [125] and inhibition ofCXCR4 activitywith the RCP168 peptide partially overcame resistance to chemotherapy (Ara-C treatment) induced in Jurkat T-ALL cells by co-culturing with BM-MSCs
Overall, these findings indicated CXCR4 inhibitors combined with chemotherapeutic agents as a potential strategy for targeting T-ALL cell/BMmicroenvironment interactions.
It was found that direct T-ALL cell interaction with CXCL12producing vascular endothelial cells was required for disease maintenance andprogression in vivo, suggesting the importance ofthe vascular niche in T-ALL pathophysiology
In addition, it has been reported that BM stromal cells up-regulated IL-8mRNA in T-ALL cells through the activity ofCXCR4
, CXCR7 is the second receptor for CXCL12 [131].A recent study has highlighted that CXCR7 mRNA was more expressed in ALL primary cells, than in acute myelogenous leukemia (AML) blasts or healthy BMmononuclear cells
Western blot analysis of ALL cell lines confirmed a much higher expression of CXCR7 in T-ALL cell lines (Jurkat and MOLT-4) when compared with B-ALL cell lines (Raji and Daudi), whereas the expression of CXCR4 was similar in both T- and B-ALL cell lines
17、（P7）Endothelial cells of the BM microenvironment blood vessels expressed high level of DLL-4 and activated specifically Notch3 signaling in T-ALL cells, thus promoting tumor escape from dormancy
18、（P9）Initial studies on relapsed B-ALL patients, aimed to better characterize the leukemic cells, led to the discovery that the expression levels of a broad spectrum of cytokines (which included IL-7, IL-10, IL-15, and IFN-γ) and their receptors were higher as compared with healthy BM samples.
BM-MSCs could also increase survival and proliferation of T-ALL cells through IL-7
19、（P10）Moreover, EVs isolated from serumdeprived MSCs, were protective, by transporting supportive miRs and promoting breast tumor growth in vivo
20、（P10）Exosomes isolated from AML cells were enriched in transcripts relevant for leukemia prognosis and stem cell niche function: FLT3, nucleophosmin 1 (NPM1), CXCR4, MMP-9, and IGF-1R. IGF-1R played an important role in stimulating induction of down-stream gene expression and changes in cell proliferation
21、（P10）. It was also demonstrated that exosomes derived fromBM-MSCswere able to promote migration ofMM cells. This was at least partially due to the up-regulation of CXCL12/CXCR4/monocyte chemoattractant protein-1 (MCP-1) axis [194]. Moreover, BM MSC-derived exosomes were able to increase MM cell survival and proliferation by up-regulating anti-apoptotic Bcl-2, and activating signaling cascades that included p38 MAPK, p53/JNK, and PI3K/Akt
21、（P10）The levels ofGal-3mRNA and protein expression in B-ALL cells were in relation with the appearance of drug-resistance.
22、（P11）For example, one potential serious drawback of inhibiting CXCL12/ CXCR4 interactions, is that leukemic cells will be massively released into the peripheral blood. While CXCR4 inhibition will drive leukemic cells out of their protective bone marrow niches and likely make them more sensitive to chemotherapy, some experts have expressed concern that mobilized leukemic cells could have the potential for infiltrating to a greater extent extra-medullary organs. Therefore, all of the currently open clinical trials examining CXCR4 inhibitors as chemosensitizing agents, are using the inhibitors in combinationwithmore than one chemotherapeutic drug, thereby increasing the probability that mobilized leukemic cells will be killed