scholarly journals SATB1 Expression Helps in Identification of the Lymphoid-Lineage-Biased Trajectory of Functionally Fluctuating Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 424-424
Author(s):  
Yukiko Doi ◽  
Takafumi Yokota ◽  
Yusuke Satoh ◽  
Tomoaki Ueda ◽  
Yasuhiro Shingai ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Research Funding ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Lineage Differentiation ◽  
Reporter Mice ◽  
Satb1 Expression ◽  
Serial Transplantation

Abstract Hematopoietic stem cells (HSCs) are purified well using a combination of surface markers. However, even highly enriched HSC fractions have heterogeneity in their self-renewal and differentiation potential. These seemingly contradictory roles are well regulated according to the changing demand for blood cells. Hence, interactions among lineage-related genes need to be set up, as their differentiation potential is restricted. However, how the functional diversity of HSCs reflects their intrinsic gene expression is not yet known. We previously identified special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, as a lymphoid-inducing gene in HSCs (Immunity 2013). SATB1 overexpression strongly enhanced lymphopoiesis from murine HSCs, whereas SATB1 deficiency caused HSC malfunctions. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent and differentiated preferentially to myeloid-erythroid lineages (Nat Immunol 2013). These results suggested that SATB1 is indispensable not only for the lymphopoietic potential but also for the normal function of HSCs. In this study, we first prepared hematological-lineage restricted SATB1 conditional knock out (cKO) mice to examine whether SATB1 is essential for normal HSC function in the adult bone marrow (BM). We crossed SATB1-flox mice with Cre-recombinase expressing mice under control of the Tie2 gene promoter, which efficiently inactivated the target gene in HSCs. Analyzing the BM in these mice, we observed a significant decrease in the number of HSCs as compared to those in their wild type (WT) littermates. Next, we collected HSCs from WT and Tie2-Cre SATB1-flox cKO mice using flow cytometry, and transplanted these CD45.2+ HSCs into CD45.1+ congenic mice. The chimerism of the transplanted cells was lower in recipients of SATB1 cKO mice-derived HSCs. Evaluation of the lymphocytic potential in a co-culture with MS5 stromal cells revealed that the output of lymphocytes from SATB1-cKO HSCs was lower than that of WT HSCs. Secondly, we generated SATB1 reporter mice in which SATB1 expression can be precisely monitored in vivo, and examined the early differentiation of HSCs. We found that the HSC fraction of adult BM consists of SATB1− and SATB1+ cells. We sorted the two types of HSCs with high purity and compared their growth and differentiation potential in vitro and in vivo. In methylcellulose colony assays, SATB1+ HSCs were less potent for producing myeloid-erythroid lineage colonies. In the co-culture with MS5 stromal cells, the output of lymphocytes from SATB1+ HSCs was more robust than that from SATB1− HSCs. RNA-sequencing data showed that the expression of many lymphocyte-related genes was upregulated in the SATB1+ HSCs compared to that in the SATB1− HSCs; however, there were no significant differences between the expression of stem cell-related genes in the two HSC types. In serial transplantation experiments, the SATB1+ HSCs produced more lymphocytic cells and fewer myeloid cells in the first recipients. Moreover, both types of HSCs could equally reconstitute the complete HSC fraction that contained SATB1− and SATB1+ cells, and successfully reconstituted lympho-hematopoiesis in the secondary recipients. In a study with SATB1-cKO mice, we found that SATB1 is indispensable for the preservation of the HSC potential for self-renewing proliferation and lymphocyte-differentiation. These results suggest that SATB1 plays a critical role for HSC integrity. With the newly generated SATB1 reporter mice, we confirmed the heterogeneity of HSCs. While the SATB1− and SATB1+ HSCs significantly differed in lineage-differentiation potential, both showed high long-term self-renewing capacity and reciprocal reconstitution in the serial transplantation. The cell dividing flow of the two HSC fractions settled in the same trajectory in the primary recipients, and then demonstrated equal ability for self-renewal and differentiation in the secondary recipients. Thus, we successfully isolated authentic lymphoid lineage-biased HSCs using SATB1 expression levels, and our results shed light on the oscillating nature of HSCs. Therefore, we conclude that the SATB1 expression demonstrates the fluctuation of HSCs with respect to the lineage-differentiation potential, and that SATB1 probably contributes to generation of chromatin loop formation, which endows HSCs with robust lymphopoietic potential. Disclosures Doi: Yakult Honsha Co.,Ltd.: Speakers Bureau. Yokota:SHIONOGI & CO., LTD.: Research Funding. Shibayama:Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kanakura:Chugai Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Toyama Chemical: Research Funding; Fujimotoseiyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Shionogi: Research Funding; Alexionpharma: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Nippon Shinyaku: Research Funding.

SATB1 Expression Marks Lymphoid-Lineage-Biased Hematopoietic Stem Cells in Mouse Bone Marrow

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2356-2356 ◽  
Author(s):  
Yukiko Doi ◽  
Takafumi Yokota ◽  
Tomohiko Ishibashi ◽  
Yusuke Satoh ◽  
Michiko Ichii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stromal Cells ◽  
System Development ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
B Lineage ◽  
Satb1 Expression ◽  
And Function

Abstract Background: Lifelong hematopoiesis is maintained by cell differentiation in which signaling pathways and transcription factors coordinately induce step-wise maturation of hematopoietic stem cells (HSCs) toward downstream effector cells. In addition, the organization of chromatin structure that creates accessible sites of target genes is also essential so as to ensure temporally and spatially adequate control of internal gene expression. Murine HSCs can be isolated with high efficiency using surface molecules including lineage-related markers, c-Kit, Sca-1, Flt3 and SLAM family proteins. However, even the highly enriched HSC fraction is still heterogeneous regarding differentiation potential, and how the HSC diversity reflects the heterogeneity of intrinsic gene-expression in HSCs is as-yet-unknown. We previously identified Special AT-rich Sequence Binding protein 1 (SATB1), a global chromatin regulator, as a lymphoid-related gene in the HSC differentiation (Satoh and Yokota et al. Immunity 2013). Indeed, SATB1 overexpression strongly enhanced both T and B lymphopoietic potential of murine HSCs whereas SATB1 deficiency caused malfunctions of HSCs in the lymphopoietic activity. Furthermore, another report showed that SATB1-deficient HSCs were less quiescent in transplanted recipients and more prone to differentiate preferentially to myeloid-erythroid lineages (Will et al. Nat Immunol 2013). These results suggested that SATB1 is likely indispensable not only for the lymphopoietic potential but also for the integrity of HSCs. Here, to better understand the mechanism how SATB1 influences homeostatic HSC functions in adult bone marrow (BM), we have developed a new mouse model in which SATB1 expression can be precisely monitored along the HSC differentiation. Methods: The Tomato gene, coding a red fluorescent protein, was knock-in to the coding region of endogenous Satb1 gene. The heterozygous SATB1/Tomato knock-in mice in which one Satb1 allele was replaced with the Tomato were used to sort HSCs in adult BM. The sorted cells were evaluated for the differentiation potential with methylcellulose colony assays and co-cultures with MS5 stromal cells. Further, the long-term reconstitution ability was evaluated by transplantation to lethally irradiated mice. To obtain transcriptome information, total RNA was isolated from SATB1/Tomato- and SATB1/Tomato+ HSCs, and then next-generation sequencing was performed. The data were analyzed with the Ingenuity Pathway Analysis software. Results: We defined Lin- Sca1+ c-KitHi (LSK) CD150+ Flt3- cells as HSCs, especially adopting FLT3- to exclude FLT3+ lymphoid-primed multipotent progenitors from our functional analyses. We found that the LSK CD150+ Flt3- fraction contains substantial number of SATB1/Tomato+ cells. While both SATB1/Tomato- and SATB1/Tomato+ HSCs produced numerous CFU-Mix and CFU-GM/G/M colonies, the latter were less potent to produce BFU-E. In co-culture with MS5 stromal cells that support B and myeloid lineages, the output of B lineage cells from SATB1+ HSCs was more robust than that of SATB1- HSCs. Upon transplantation, enhanced B-lineage engraftment was observed in the SATB1+ HSC-transplanted recipients. Interestingly, while the two types of HSCs showed obvious difference in the differentiation potential toward lymphoid or myeloid lineage, both HSCs reconstituted the LSK CD150+ Flt3- fraction that similarly contained SATB1/Tomato- and SATB1/Tomato+ cells. With the RNA-sequencing data of SATB1- and SATB1+ HSCs, biological pathway analyses revealed that the "Hematological System Development and Function" pathway was remarkably up-regulated in the SATB1+ HSCs. Among subcategories of the "Hematological System Development and Function" pathway, the "quantity of lymphocytes" pathway was increased whereas "quantity of myeloid cells" and "quantity of granulocytes" pathways were decreased. Conclusion: We have developed a new mouse system that can be used to identify and isolate viable lymphoid-biased HSCs in the most primitive hematopoietic cell fraction of adult BM. While the SATB1- and SATB1+ HSCs differ genetically and functionally, both subtypes have displayed a self-renewal activity with mutual interconversion in transplanted recipients. These findings suggest that functional heterogeneity and variability within the HSC population is, at least in part, a manifestation of SATB1 expression. Disclosures Yokota: SHIONOGI & CO., LTD.: Research Funding.

scholarly journals Pertussis toxin–sensitive G proteins regulate lymphoid lineage specification in multipotent hematopoietic progenitors

Blood ◽  
2009 ◽  
Vol 113 (23) ◽  
pp. 5757-5764 ◽  
Author(s):  
Anne Y. Lai ◽  
Akiko Watanabe ◽  
Tommy O'Brien ◽  
Motonari Kondo
Keyword(s):  
Bone Marrow ◽  
Pertussis Toxin ◽  
Distal Region ◽  
Lineage Specification ◽  
Differentiation Potential ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage

Abstract Lymphoid and myeloid lineage segregation is a major developmental step during early hematopoiesis from hematopoietic stem cells. It is not clear, however, whether multipotent progenitors (MPPs) adopt a lymphoid or myeloid fate through stochastic mechanisms, or whether this process can be regulated by extracellular stimuli. In this study, we show that lymphoid lineage specification occurs in MPPs before lymphoid lineage priming, during which MPPs migrate from the proximal to the distal region relative to the endosteum of the bone marrow. Lymphoid-specified MPPs have low myeloid differentiation potential in vivo, but potently differentiate into myeloid cells in vitro. When treated with pertussis toxin, an inhibitor of G protein–coupled receptor signaling, lymphoid-specified MPPs regain in vivo myeloid potential, and their localization is dispersed in the bone marrow. These results clearly demonstrate that specific microenvironments that favorably support lymphoid or myeloid lineage development exist at structurally distinct regions in the bone marrow.

scholarly journals Novel Lymphoid Pathway of Human Plasmacytoid and Conventional Dendritic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4998-4998
Author(s):  
Keiki Nagaharu ◽  
Kohshi Ohishi ◽  
Naoyuki Katayama
Keyword(s):  
Dendritic Cells ◽  
Nk Cells ◽  
Stromal Cells ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Multipotent Progenitors ◽  
Lymphoid Progenitors

[Introduction] Dendritic cells (DCs) play a central role in initiation and regulation of immune response. Human plasmacytoid DCs (pDCs) as well as conventional DCs (cDCs) were shown to differentiate from multi-lymphoid progenitors (MLPs) as well as myeloid progenitors via common DC progenitors. However, lymphoid pathway of DCs remained clarified. Here we investigated lymphoid origin of DCs, using a novel co-culture system which supports differentiation of various lineages of lymphoid and DCs (Br J Haematol. 157:674, 2012; J Immunol. 199:2343, 2017). [Methods and Results] CD34+CD38-CD45RA-CD10-CD7- human hematopoietic stem/progenitor cells (HSPCs) and various lymphoid progenitors including CD34+CD38-CD45RA+CD10-CD7-lymphoid-primed multipotent progenitors (LMPPs), CD34+CD38-CD45RA+CD10+ MLPs,and CD34+CD38+CD45RA+CD10+CD7-CD19- (10SPs), generally though as B/NK progenitors, were isolated from cord blood and cocultured with telomerized human stromal cells with SCF, flt3L, TPO, and GM-CSF. After 17 to 21 days, generation of CD45RA+CD19+ proB, CD56+CD3- NK cells, HLA-DR+IL-3RhighCD303+CD304+pDCs, and CD1c+cDCs was assessed. We observed that not only immature HSPCs, LMPPs and MLPs but also 10SPs gave rise to pDC and cDCs in addition to proB, NK cells. 10SPs were found to be subdivided by expression pattern of c-kit and IL-7 receptor (R), and similarly cocultured with stromal cells. pDCs, cDCs, proB, and NK cells were generated from c-kit+IL-7R- fraction, while pDC, cDCs, and proB, but few or no NK cells were generated from c-kit+IL-7R+ fractions. c-kit-IL-7R+ fraction produced a significantly lower number of cells than c-kit+IL-7R+or- fractions and mainly differentiated into proB cells. By single cell assay of 10SPs, progenitors with differentiation potential for pDC, cDC, and/or proB were detected. These data revealed a differentiation pathway of pDCs and cDCs from relatively mature lymphoid progenitors and suggested the presence of DC and B common progenitors. [Summary] The present study uncovered a closed relationship between B-lymphoid and DC differentiation pathway. We are further attempting to delineate their relationship and differentiation process toward pDCs, cDCs, and proB. Disclosures Nagaharu: kyowa hakko kirin: Research Funding; Astellas Pharma: Research Funding; Nippon Shinyaku: Research Funding; Ono Pharmaceutical: Research Funding. Ohishi:kyowa hakko kirin: Research Funding; Nippon Shinyaku: Research Funding; Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding. Katayama:Sysmex: Honoraria; Taisho Toyama Pharma: Honoraria; Celgene: Honoraria; Pfizer: Honoraria; Alexion Pharmaceuticals: Honoraria; Chugai: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Ono Pharmaceutical: Research Funding; Novo Nordisk: Honoraria; Shionogi Pharmaceutical: Honoraria; Shire: Honoraria; Novartis: Honoraria; Astellas Pharma: Honoraria, Research Funding; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria; kyowa hakko kirin: Honoraria, Research Funding.

scholarly journals Asymmetrical lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors

2006 ◽  
Vol 203 (8) ◽  
pp. 1867-1873 ◽  
Author(s):  
Anne Y. Lai ◽  
Motonari Kondo
Keyword(s):  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Differentiation Potential ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Lineage Differentiation ◽  
Multipotent Progenitors ◽  
Sequential Loss ◽  
Cell Adhesion Molecule 1

The mechanism of lineage commitment from hematopoietic stem cells (HSCs) is not well understood. Although commitment to either the lymphoid or the myeloid lineage is popularly viewed as the first step of lineage restriction from HSCs, this model of hematopoietic differentiation has recently been challenged. The previous identification of multipotent progenitors (MPPs) that can produce lymphocytes and granulocyte/macrophages (GMs) but lacks erythroid differentiation ability suggests the existence of an alternative HSC differentiation program. Contribution to different hematopoietic lineages by these MPPs under physiological conditions, however, has not been carefully examined. In this study, we performed a refined characterization of MPPs by subfractionating three distinct subsets based on Flt3 and vascular cell adhesion molecule 1 expression. These MPP subsets differ in their ability to give rise to erythroid and GM lineage cells but are equally potent in lymphoid lineage differentiation in vivo. The developmental hierarchy of these MPP subsets demonstrates the sequential loss of erythroid and then GM differentiation potential during early hematopoiesis. Our results suggest that the first step of lineage commitment from HSCs is not simply a selection between the lymphoid and the myeloid lineage.

Unexpectedly High Self-Renewal Capacity Demonstrable by Serial Transplantation Is a Property of a Subset of Normal Adult Mouse Hematopoietic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1189-1189
Author(s):  
Brad Dykstra ◽  
Naoyuki Uchida ◽  
Melisa Hamilton ◽  
Merete Kristiansen ◽  
Kristin Lyons ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Single Cell ◽  
Marrow Cell ◽  
Normal Adult ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Definitive Evidence ◽  
Serial Transplantation

Abstract The hallmark of a hematopoietic stem cell (HSC) is its capacity to sustain production of daughter cells with the same multi-lineage differentiation potential for the lifetime of the organism. Definitive evidence of HSC self-renewal requires the demonstration that daughter HSCs have been generated from the division of a single cell. This was first demonstrated by the detection of clones with the same proviral inserts in multiple secondary recipients of retrovirally-marked HSCs initially expanded in primary mice. Later, this approach showed that HSC self-renewal occurs in longterm marrow cultures. More recently, HSC self-renewal divisions were demonstrated by in vivo assessment of the progeny of highly purified (lin- Rhodamine-123- SP) HSCs stimulated to divide in single-cell cultures containing recombinant growth factors. We now describe the remarkable self-renewal activity that a proportion of HSCs can display in vivo when subjected to serial transplantation. In 7/25 (28%) sublethally irradiated B6-W41/W41-Ly-5.2 mice engrafted with a single lin- Rho- SP Ly5.1 bone marrow cell, the majority of the circulating WBCs (all lineages) 6 months later were Ly-5.1. 1.4-5.0% of the Ly-5.1 cells present in the marrow of 3 of these mice was then transplanted into 1 or 2 sublethally irradiated secondary B6-W41/W41-Ly-5.2 recipients. Six months later, at least one of these secondary recipients of cells from each of the 3 primary mice contained 1-79% Ly5.1 WBCs including all lineages. Ly5.1 cells from only one of the 3 clones regenerated in these secondary recipients repopulated tertiary mice, reflecting the heterogeneity of HSC self-renewal activity. Ly-5.1 marrow cells obtained 6 months post-transplant from the repopulated tertiary mice (1–4% of all the Ly-5.1 cells) were transplanted into 4th and 5th generation recipients at further 6-month intervals. Three of 6 of the 4th generation recipients and 1 of 4 of the 5th generation recipients were significantly repopulated (8–20% Ly5.1 cells in the blood after 12–16 weeks ). However, the proportions of Ly5.1 myeloid cells were low in one of the 3 repopulated 4th generation recipients and in the single repopulated 5th generation recipient (0.5–1% of the Ly5.1 clone in the blood after 12–16 weeks, compared to >5% in all other repopulated mice). Assuming that these latter 2 mice received at least one HSC, and that HSCs are randomly distributed throughout the entire bone marrow mass, it could be calculated from the serial transplant data that the most prolific original HSC transplanted had the ability to produce more than 780,000 daughter HSCs through the execution of at least 19 symmetric self-renewal divisions (and more if not all self-renewal divisions were symmetric). If the 2 mice with low myeloid repopulation are not included, the same calculation indicates an output capacity of 28,000 daughter HSCs requiring at least 14 symmetric self-renewal divisions. In addition, at least 20 subsequent divisions would need to have occurred to account for the >1 million donor-derived WBCs produced in each 5th generation mouse. These results provide the first data quantifying the extraordinary self-renewal and proliferative capacity of a subset of the HSCs in the marrow of normal adult mice.

Lymphoid Lineage Restriction in Hematopoietic Stem Cells through Their First Division.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 368-368
Author(s):  
Hideo Ema ◽  
Makoto Kaneda ◽  
Azusa Maeda ◽  
Hina Takano ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Asymmetric Division ◽  
Lineage Commitment ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Lymphoid Lineage ◽  
Lineage Differentiation ◽  
B Lineage

Abstract Little is known of how hematopoietic stem cells (HSCs) differentiate. We have previously suggested that particular myeloid lineages, such as a neutrophil/macrophage (nm) lineage, arise asymmetrically through their first division. In this study, we asked whether lymphoid lineage restriction similarly takes place at the level of HSCs. CD34−c-Kit+Sca-1+lineage−(KSL) cells and CD34+KSL cells, respectively, highly enriched for long-term and short-term repopulating cells, were isolated from bone marrow (BM) of B6 mice by FACS. These cells were subjected to in vitro colony assay and in vivo repopulation assay with use of Rag-2-deficient BM cells, instead of normal BM cells, as competitor cells to facilitate the detection of lymphoid lineage reconstitution. Single CD34−KSL cells were allowed to divide once in the presence of SCF+TPO or SCF+IL-3 under serum-free culture condition. Resultant paired daughter (PD) cells were subsequently separated by micromanipulation and individually transplanted into lethally irradiated mice. Recipient mice were analyzed 7 weeks after transplantation to detect myeloid (My), B-lymphoid (B), and T-lymphoid (T) lineage repopulation by single test donor cells. When single CD34−KSL cells were injected into lethally irradiated mice, about 20% of the cells were detected as MyBT lineage repopulating cells. Interestingly, about 25% of the cells were detected as MyT lineage repopulating cells without detectable level of B lineage reconstitution. Limiting dilution analysis of CD34+KSL cells estimated the frequency of B lineage repopulating cells as close to 1 in 10 cells and that of T lineage repopulating cells as about 1 in 80 cells. Because most colony forming cells among CD34+KSL cells exclusively gave rise to nm lineage, it was assumed that the differentiation potential of CD34+KSL cells was mostly restricted to nm and B lineages. These data suggest more MyT repopulating cells present in CD34− fraction than in CD34+ fraction and, in contrast, more MyB repopulating cells present in CD34+ fraction than in CD34− fraction among KSL cells. On the other hand, My lineage repopulation was observed in about 60% of the PD cells. B and T, B, or T lineage differentiation potential was also detected in approximately 8%, 8%, or 40% of the PD cells with My lineage repopulating activity when generated in the presence of SCF+TPO and in 2%, 5%, or 2% of the cells when generated in the presence of SCF+IL-3. As compared with freshly isolated CD34−KSL cells, T, but not B lineage differentiation potential was well maintained in PD cells by SCF+TPO whereas either B or T lineage differentiation potential was hardly maintained in PD cells by SCF+IL-3. These data indicate the instructive role of cytokines in the restriction of lymphoid potential in HSCs. MyT lineage repopulating cells appeared to be directly generated from MyBT stem cells via their asymmetric division. Taken together, we conclude that asymmetric division of HSCs results in their lymphoid lineage restriction and that T lineage commitment takes place at the level of HSCs, independent of and prior to B lineage commitment which occurs at later stages of differentiation. We propose a novel differentiation model of HSCs which challenges the CLP and CMP based model.

scholarly journals Tolerance to Bone Marrow Transplantation: Do Mesenchymal Stromal Cells Still Have a Future for Acute or Chronic GvHD?

2020 ◽  
Vol 11 ◽  
Author(s):  
Martino Introna ◽  
Josée Golay
Keyword(s):  
Clinical Trials ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Pathological Condition ◽  
Statistical Significance ◽  
Chronic Gvhd ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Mesenchymal Stromal Cells (MSCs) are fibroblast-like cells of mesodermal origin present in many tissues and which have the potential to differentiate to osteoblasts, adipocytes and chondroblasts. They also have a clear immunosuppressive and tissue regeneration potential. Indeed, the initial classification of MSCs as pluripotent stem cells, has turned into their identification as stromal progenitors. Due to the relatively simple procedures available to expand in vitro large numbers of GMP grade MSCs from a variety of different tissues, many clinical trials have tested their therapeutic potential in vivo. One pathological condition where MSCs have been quite extensively tested is steroid resistant (SR) graft versus host disease (GvHD), a devastating condition that may occur in acute or chronic form following allogeneic hematopoietic stem cell transplantation. The clinical and experimental results obtained have outlined a possible efficacy of MSCs, but unfortunately statistical significance in clinical studies has only rarely been reached and effects have been relatively limited in most cases. Nonetheless, the extremely complex pathogenetic mechanisms at the basis of GvHD, the fact that studies have been conducted often in patients who had been previously treated with multiple lines of therapy, the variable MSC doses and schedules administered in different trials, the lack of validated potency assays and clear biomarkers, the difference in MSC sources and production methods may have been major factors for this lack of clear efficacy in vivo. The heterogeneity of MSCs and their different stromal differentiation potential and biological activity may be better understood through more refined single cell sequencing and proteomic studies, where either an “anti-inflammatory” or a more “immunosuppressive” profile can be identified. We summarize the pathogenic mechanisms of acute and chronic GvHD and the role for MSCs. We suggest that systematic controlled clinical trials still need to be conducted in the most promising clinical settings, using better characterized cells and measuring efficacy with specific biomarkers, before strong conclusions can be drawn about the therapeutic potential of these cells in this context. The same analysis should be applied to other inflammatory, immune or degenerative diseases where MSCs may have a therapeutic potential.

scholarly journals Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marie-Theresa Weickert ◽  
Judith S. Hecker ◽  
Michèle C. Buck ◽  
Christina Schreck ◽  
Jennifer Rivière ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Fate ◽  
Stromal Cells ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Bone Marrow Niche ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Bm Niche

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

scholarly journals Mesenchymal stromal cells can be applied to red blood cells storage as a kind of cellular additive

2017 ◽  
Vol 37 (5) ◽  
Author(s):  
Yaozhen Chen ◽  
Jing Zhang ◽  
Shunli Gu ◽  
Dandan Yin ◽  
Qunxing An ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Storage Duration ◽  
Hematopoietic Stem ◽  
Diminished Capacity ◽  
Blood Banks ◽  
Citrate Phosphate

During storage in blood banks, red blood cells (RBCs) undergo the mechanical and metabolic damage, which may lead to the diminished capacity to deliver oxygen. At high altitude regions, the above-mentioned damage may get worse. Thus, more attention should be paid to preserve RBCs when these components need transfer from plain to plateau regions. Recently, we found that mesenchymal stromal cells (MSCs) could rescue from anemia, and MSCs have been demonstrated in hematopoietic stem cells (HSCs) transplantation to reconstitute hematopoiesis in vivo by us. Considering the functions and advantages of MSCs mentioned above, we are trying to find out whether they are helpful to RBCs in storage duration at high altitudes. In the present study, we first found that mice MSCs could be preserved in citrate phosphate dextrose adenine-1 (CPDA-1) at 4 ± 2°C for 14 days, and still maintained great viability, even at plateau region. Thus, we attempted to use MSCs as an available supplement to decrease RBCs lesion during storage. We found that MSCs were helpful to support RBCs to maintain biochemical parameters and kept RBCs function well on relieving anemia in an acute hemolytic murine model. Therefore, our investigation developed a method to get a better storage of RBCs through adding MSCs, which may be applied in RBCs storage as a kind of cellular additive into preservation solution.

scholarly journals Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Gordon G. L. Wong ◽  
Gabriela Krivdova ◽  
Olga I. Gan ◽  
Jessica L. McLeod ◽  
John E. Dick ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Lineage Differentiation ◽  
Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

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