Changes in the Multipotent Stromal Mesenchymal Cells from the Bone Marrow of the Patients with Hematological Diseases in Debut and after the Treatment

Introduction. Stromal microenvironment of the bone marrow (BM) is essential for normal hematopoiesis; the very same cells are involved in the interaction with the leukemic stem cells. The aim of the study was to reveal the alterations in stromal microenvironment of patients in debut and after the therapy using multipotent mesenchymal stromal cells (MSC) as a model. Methods. MSC of patients with acute myeloid leukemia (AML, N=32), acute lymphoblastic leukemia (ALL, N=20), chronic myeloid leukemia (CML, N=19), and diffuse large B-cell lymphoma without BM involvement (DLBCL, N=17) were isolated by standard method from the patients' BM. Each BM sample was acquired during diagnostic aspiration after the informed signed consent was obtained from the patient. Groups of BM donors comparable by age and gender were used as controls for each nosology. Gene expression was analyzed with real-time RT-PCR. The significance of differences was evaluated with Mann-Whitney U-test. Results. The results of gene expression analysis are summarized in Table. The expression of genes regulating hematopoietic stem and precursor cells (JAG1, LIF, IL6) was significantly upregulated in MSC of the patients in debut, except for DLBCL. The latter was characterized with upregulation of osteogenic marker gene SPP1 and downregulation of FGFR1 gene. The upregulation of the expression of genes regulating proliferation of stromal cells (PDGFRA, FGFR1) and adipogenic marker gene (PPARG) was common for AML and CML. Both acute leukemias were characterized by the upregulation of genes associated with inflammation and regulation of hematopoietic precursors (CSF1, IL1B, IL1BR1) and by the downregulation of chondrogenic differentiation marker gene (SOX9). CML and DLBCL demonstrated the upregulation of FGFR2. BM of the DLBCL patients did not contain any malignant cells; nevertheless, stromal precursors from the BM were significantly affected. This indicates the distant effects of DLBCL malignant cells on the patients' BM. Myeloid malignancies seem to affect MSC more profoundly then lymphoid ones. Effect of leukemic cells on stromal microenvironment in case of myeloid leukemia was more pronounced. The treatment significantly affected gene expression in MSC of patients. In all studied nosologies the IL6 gene expression was upregulated, which may reflect the inflammation processes ongoing in the organism. The expression of LIF was upregulated and ICAM1, downregulated in MSCs of AML, ALL, and CML patients. In the MSC of patients with AML, who had received the highest doses of cytostatic drugs to achieve remission, a significant decrease in the expression of most studied genes was found. In patients with ALL with long-term continuing treatment in combination with lower doses of drugs, IL1B expression was increased, while the decrease in expression was detected for a number of genes regulating hematopoietic stem cells (SDF1, TGFB1), differentiation and proliferation (SOX9, FGFR1, FGFR2). Treatment of CML patients is based on tyrosine kinase inhibitors in doses designed for long-term use, and is less damaging for MSC. The upregulation of TGFB1, SOX9, PDGFRA genes and downregulation of IL1B gene was revealed. MCS of DLBCL patients, unlike the other samples, were analyzed after the end of treatment. Nevertheless, significant upregulation of IL8 and FGFR2 genes was found. Thus, both the malignant cells and chemotherapy affect stromal precursor cells. The changes are not transient; they are preserved for a few months at least. MSCs comprise only a minor subpopulation in the BM in vivo. When expanded in vitro, they demonstrate significant changes between groups of patients and healthy donors. Conclusions. Leukemia cells adapt the stromal microenvironment. With different leukemia, the same changes are observed in the expression of genes in MSC. MSC of patients with acute forms have a lot of changes which coincide among these two diseases. MSC of AML patients are most affected both in debut and after the therapy. Treatment depends on the nosology and in varying degrees changes the MSC. This work was supported by the Russian Foundation for Basic Research, project no. 17-00-00170. Disclosures Chelysheva: Novartis: Consultancy, Honoraria; Fusion Pharma: Consultancy. Shukhov:Novartis: Consultancy; Pfizer: Consultancy. Turkina:Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; fusion pharma: Consultancy.

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Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD–induced myeloproliferation

Journal of Experimental Medicine ◽

10.1084/jem.20161418 ◽

2017 ◽

Vol 214 (7) ◽

pp. 2005-2021 ◽

Cited By ~ 26

Author(s):

Adam J. Mead ◽

Wen Hao Neo ◽

Nikolaos Barkas ◽

Sahoko Matsuoka ◽

Alice Giustacchini ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Stromal Cells ◽

Negative Regulator ◽

Bone Marrow Niche ◽

Strong Negative Correlation ◽

Hematopoietic Stem ◽

A Cell ◽

Tandem Duplications

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD–driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

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Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Blood ◽

10.1182/blood-2021-151354 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1131-1131

Author(s):

Alena I. Dorofeeva ◽

Irina N. Shipounova ◽

Nina J. Drize ◽

Anton V. Luchkin ◽

Zalina T. Fidarova ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Aplastic Anemia ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Multipotent Mesenchymal Stromal Cells ◽

Hematopoietic Stem ◽

Stromal Microenvironment ◽

The Difference ◽

Pnh Clone

Abstract Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions. We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes. The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p<0.05 when Student's unpaired t-test or Mann-Whitney test was applied. MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA. Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun. Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression. The work is supported by the RFBR, project 19-015-00280. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood.v84.1.74.74 ◽

1994 ◽

Vol 84 (1) ◽

pp. 74-83 ◽

Cited By ~ 36

Author(s):

SJ Szilvassy ◽

S Cory

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Bone Marrow Cells ◽

High Titer ◽

Marker Gene ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

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Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation

Blood ◽

10.1182/blood.v95.11.3620.011k02_3620_3627 ◽

2000 ◽

Vol 95 (11) ◽

pp. 3620-3627 ◽

Cited By ~ 1

Author(s):

Graça Almeida-Porada ◽

Christopher D. Porada ◽

Nam Tran ◽

Esmail D. Zanjani

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Stromal Cell ◽

In Utero ◽

Human Cells ◽

Fetal Sheep ◽

Hematopoietic Stem ◽

Donor Cells ◽

Hsc Transplantation

Both in utero and postnatal hematopoietic stem cell (HSC) transplantation would benefit from the development of approaches that produce increased levels of engraftment or a reduction in the period of time required for reconstitution. We used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantation. We hypothesized that providing a more suitable microenvironment in the form of human stromal cell progenitors simultaneously with the transplanted human HSC would result in higher rates of engraftment or differentiation of the human cells in this xenogeneic model. The results presented here demonstrate that the cotransplantation of both autologous and allogeneic human bone marrow-derived stromal cell progenitors resulted in an enhancement of long-term engraftment of human cells in the bone marrow of the chimeric animals and in earlier and higher levels of donor cells in circulation both during gestation and after birth. By using marked stromal cells, we have also demonstrated that injected stromal cells alone engraft and remain functional within the sheep hematopoietic microenvironment. Application of this method to clinical HSC transplantation could potentially lead to increased levels of long-term engraftment, a reduction in the time for hematopoietic reconstitution, and a means of delivery of foreign genes to the hematopoietic system.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v1 ◽

2019 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow, which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at the mRNA level in the cells of the hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization.Results: The results found that the bone marrow cells of patients with acute myeloid leukemia (AML) show over expression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed the positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v4 ◽

2020 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression.Methods: The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. Results: The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML.Conclusions: Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

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Different regulation of PARP1, PARP2, PARP3 and TRPM2 genes expression in acute myeloid leukemia cells

10.21203/rs.2.19004/v2 ◽

2020 ◽

Author(s):

Paulina Gil-Kulik ◽

Ewa Dudzińska ◽

Elżbieta Radzikowska-Büchner ◽

Joanna Wawer ◽

Mariusz Jojczuk ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Mrna Level ◽

Hematopoietic Stem ◽

Genes Expression ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a heterogenic lethal disorder characterized by the accumulation of abnormal myeloid progenitor cells in the bone marrow which results in hematopoietic failure. Despite various efforts in detection and treatment, many patients with AML die of this cancer. That is why it is important to develop novel therapeutic options, employing strategic target genes involved in apoptosis and tumor progression. Methods The aim of the study was to evaluate PARP1, PARP2, PARP3, and TRPM2 gene expression at mRNA level using qPCR method in the cells of hematopoietic system of the bone marrow in patients with acute myeloid leukemia, bone marrow collected from healthy patients, peripheral blood of healthy individuals, and hematopoietic stem cells from the peripheral blood after mobilization. Results The results found that the bone marrow cells of the patients with acute myeloid leukemia (AML) show overexpression of PARP1 and PARP2 genes and decreased TRPM2 gene expression. In the hematopoietic stem cells derived from the normal marrow and peripheral blood after mobilization, the opposite situation was observed, i.e. TRPM2 gene showed increased expression while PARP1 and PARP2 gene expression was reduced. We observed positive correlations between PARP1, PARP2, PARP3, and TRPM2 genes expression in the group of mature mononuclear cells derived from the peripheral blood and in the group of bone marrow-derived cells. In AML cells significant correlations were not observed between the expression of the examined genes. In addition, we observed that the reduced expression of TRPM2 and overexpression of PARP1 are associated with a shorter overall survival of patients, indicating the prognostic significance of these genes expression in AML. Conclusions Our research suggests that in physiological conditions in the cells of the hematopoietic system there is mutual positive regulation of PARP1, PARP2, PARP3, and TRPM2 genes expression. PARP1, PARP2, and TRPM2 genes at mRNA level deregulate in acute myeloid leukemia cells.

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Intrinsic Type 1 Interferon (IFN1) Profile of Uncultured Human Bone Marrow CD45lowCD271+ Multipotential Stromal Cells (BM-MSCs): The Impact of Donor Age, Culture Expansion and IFNα and IFNβ Stimulation

Biomedicines ◽

10.3390/biomedicines8070214 ◽

2020 ◽

Vol 8 (7) ◽

pp. 214

Author(s):

Payal Ganguly ◽

Agata Burska ◽

Charlotte Davis ◽

Jehan J. El-Jawhari ◽

Peter V. Giannoudis ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Stromal Cells ◽

Gene Expression Signature ◽

Proliferative Potential ◽

Donor Age ◽

Hematopoietic Stem ◽

Type 1 Interferon ◽

The Impact

Skeletal aging is associated with reduced proliferative potential of bone marrow (BM) multipotential stromal cells (MSCs). Recent data suggest the involvement of type 1 interferon (IFN1) signalling in hematopoietic stem cell (HSC) senescence. Considering that BM-HSCs and BM-MSCs share the same BM niche, we investigated IFN1 expression profile in human BM-MSCs in relation to donor age, culture-expansion and IFN1 (α and β) stimulation. Fluorescence-activated cell sorting was used to purify uncultured BM-MSCs from younger (19–41, n = 6) and older (59–89, n = 6) donors based on the CD45lowCD271+ phenotype, and hematopoietic-lineage cells (BM-HLCs, CD45+CD271−) were used as controls. Gene expression was analysed using integrated circuits arrays in sorted fractions as well as cultured/stimulated BM-MSCs and Y201/Y202 immortalised cell lines. IFN1 stimulation led to BM-MSC growth arrest and upregulation of many IFN1-stimulated genes (ISGs), with IFNβ demonstrating stronger effects. Uncultured MSCs were characterised by a moderate-level ISG expression similar to Y201 cells. Age-related changes in ISG expression were negligible in BM-MSCs compared to BM-HLCs, and intracellular reactive oxygen species (ROS) levels in BM-MSCs did not significantly correlate with donor age. Antiaging genes Klotho and SIRT6 correlated with more ISGs in BM-MSCs than in BM-HLCs. In patients with osteoarthritis (OA), BM-MSCs expressed considerably lower levels of several ISGs, indicating that their IFN1 signature is affected in a pathological condition. In summary, BM-MSCs possess homeostatic IFN1 gene expression signature in health, which is sensitive to in vitro culture and external IFN1 stimulation. IFN signalling may facilitate in vivo BM-MSC responses to DNA damage and combating senescence and aberrant immune activation.

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

Blood ◽

10.1182/blood.v126.23.2356.2356 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2356-2356 ◽

Cited By ~ 1

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.

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BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood-2009-04-215376 ◽

2010 ◽

Vol 115 (16) ◽

pp. 3185-3195 ◽

Cited By ~ 64

Author(s):

Mirle Schemionek ◽

Christian Elling ◽

Ulrich Steidl ◽

Nicole Bäumer ◽

Ashley Hamilton ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Multipotent Progenitor Cells

Abstract In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

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