Connexin-43 Regulates the Cell Cycle Entry of Hematopoietic Stem Cells within the Stem Cell Niche.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1500-1500
Author(s):  
Daniel Gonzalez-Nieto ◽  
Gabriel Ghiaur ◽  
Lina Li ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Marked Reduction ◽  
Connexin 43 ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 1500 Poster Board I-523 Bone marrow (BM) osteoblasts and stromal (O/S) cells are crucial in the establishment of the hematopoietic niches in the BM. Connexin 43 (Cx43) is expressed by BM stromal cells and by hematopoietic stem cells and progenitors (HSC/P) and is overexpressed in the BM endosteal space upon administration of chemotherapy or radiotherapy. We have previously reported that Cx43 is critical in fetal liver and in BM hematopoiesis. Since Cx43 is expressed by both HSC and the hematopoietic microenvironment, we dissected out the cellular mechanisms responsible for Cx43 function in the BM. We analyzed the hematopoiesis of mice deficient in Cx43 in the O/S cells (Collagen 1α-Creflox/flox; O/S-Cx43-deficient) or in the hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). Upon basal conditions, analysis of the HSC compartment of H-Cx43-deficient mice showed a ∼30% decreased content of immunophenotypically defined long-term HSC (LT-HSC) in BM of H-Cx43KO mice compared with their WT littermates, whereas there was not significant variation in the ST-HSC population content. The reduced LT-HSC population in H-Cx43KO mice was associated with a modest increased quiescence (∼12% increase of LT-HSC in G0). Interestingly, the expression of cyclin D1 and p21cip1 in the H-Cx43KO LT-HSC were 50% reduced and 4-fold increased, respectively, suggesting a decreased ability to enter cell cycle. While we found no significant engraftment difference in primary recipients of competitive repopulation assays, we found a marked reduction (>50%) in the engraftment ability of LT-HSC Cx43-deficient cells when transplanted into secondary recipients. When submitted to stress by 5-fluorouracil (5-FU) administration, H-Cx43KO mice showed a severely decreased hematopoietic recovery of peripheral blood (PB) counts for neutrophils and platelets accompanied with a marked reduction in the BM cellularity and hematopoietic progenitor content on day +14 after treatment. This defect was associated with a dramatic decreased (∼75 %) in the proliferation of the Cx43-deficient, LT-HSC population by 48 hours post-5-FU administration and a relative decrease of the expansion of the ST-HSC/MPP pool as early as 6 days post-5-FU administration. Interestingly, O/S-Cx43-deficient mice also showed severely delayed hematological recovery after 5-FU administration, with reduction in cellularity and hematopoietic progenitor content, suggesting that the increased hematopoietic toxicity induced by 5-FU in the context of Cx43 deficiency may depend on HSC-to-O/S Cx43 homotypic communication. This communication would be responsible of control of the G1 restriction checkpoint in LT-HSC. In summary, our findings suggest that Cx43 expression plays a crucial role controlling the LT-HSC pool size and fitness in response to stress. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Deficiency of Bone Marrow Stromal Cx43 Expression Induces Hematopoietic Progenitor Homing Deficiency and Cell-Cycle Arrest of Hematopoietic Stem Cells and Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 349-349
Author(s):  
Lina Li ◽  
Cynthia A. Presley ◽  
Bryan Kastl ◽  
Jose A. Cancelas
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Chimeric Mice ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Cx43 Expression ◽  
Hematopoietic Stem ◽  
M Phase ◽  
Deficient Mice

Abstract Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be critical in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. BM cells are directly connected through gap junctions (GJs) which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM OS cells. Multiple osteogenic defects have been reported in human Cx43 mutations and Cx43 has been shown to be essential in controlling osteoblast functions. Due to the perinatal death of Cx43 germline null mice, an interferon-inducible, conditional genetic approach (Mx1-Cre), expressed by both hematopoietic and stromal BM cells, was used to study the role of Cx43 in stem cell function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration (Presley C, et al., Cell Comm. Adh., 2005). Here, we observed that after 5-FU administration, Cx43 expression is predominantly located in the endosteum. To study the role of stroma-dependent Cx43 in hematopoiesis, we developed hematopoietic chimeras by BM transplantation of wild-type Cx43 HSC into stromal Cx43-deficient mice. Stromal Cx43 deficiency induced a severe impairment of blood cell formation during the recovery phase after 5-FU administration compared to stromal Mx1-Cre-Tg wild-type controls (Table 1), as well as a significant decrease in BM cellularity (~60% reduction) and progenitor cell content (~83% reduction). Cell cycle analysis of 5-FU-treated BM progenitors from stromal Cx43-deficient mice showed an S-phase arrest (S phase: 63.5%; G2/M phase: <1%) compared to wild-type chimeric mice (S phase: 38.6%, G2/M phase: 7.8%, p=0.01) suggesting a cell division blockade. Unlike Cx43-deficient primary mice, a differentiation arrest at the HSC compartment was observed in 5-FU-treated, stromal Cx43-deficient mice, since the content of competitive repopulating units (CRU) at 1 month, of 14-day post-5-FU BM of stromal Cx43-deficient mice was increased (27.7 ± 0.67) compared to recipients of HSC from stromal wild-type counterparts (26.5 ± 0.92 CRU, p < 0.01). Interestingly, wild-type hematopoietic progenitor homing in stromal Cx43-deficient BM was severely impaired with respect to wild-type BM (5.1% vs10.4 %, respectively, p < 0.01), while hematopoietic Cx43-deficient BM progenitors normally homed into the BM, suggesting a differential role for Cx43 in stromal and HSC. In conclusion, expression of Cx43 in osteoblasts and stromal cells appears to play a crucial role in the regulation of HSC homing in BM and hematopoietic regeneration after chemotherapy. Peripheral blood counts of WT and stromal Cx43-deficient chimeric mice after 5-FU administration (150 mg/Kg) Neutrophil counts (×10e9/L) Reticulocyte count (%) Day post-5-FU WT Cx43-deficient WT Cx43-deficient * p < 0.05 Day +8 2.89 ± 0.06 0.81 ± 0.02* 2.0 ± 0.6 3.0 ± 0.9 Day +11 9.11 ± 2.5 3.13 ± 0.8* 6.1 ± 0.6 2.7 ± 0.3* Day +14 6.22 ± 5.7 7.58 ± 8.2 7.5 ± 0.5 2.5 ± 0.5*

Connexin-43 Expression Regulates the Migration of Hematopoietic Stem Cells and Progenitors towards and From Bone Marrow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 562-562
Author(s):  
Daniel Gonzalez-Nieto ◽  
Kyung-Hee Chang ◽  
Anja Koehler ◽  
Jorden Arnett ◽  
Susan Dunn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Connexin 43 ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Hematopoietic Microenvironment ◽  
Deficient Mice

Abstract Abstract 562 In the bone marrow (BM) cavity, the migratory traffic of hematopoietic stem cells and progenitors (HSC/P) from the endosteal niches to circulation and viceversa depends on their response to chemokine gradients and interaction with endothelial and mesenchymal pre-osteoblastic cells located at the endosteal niches, forming the hematopoietic microenvironment (HM). Several lines of evidence have pointed out the possible role of the gap junction-forming protein connexin-43 (Cx43) in the control of stem cell and progenitor migration. Our group previously demonstrated that Cx43 expression in the hematopoietic microenvironment (HM) is critical in the fetal liver and BM hematopoietic regeneration after administration of 5-fluorouracil (5-FU) and other investigators have shown that Cx43 is crucial controlling the migration of neural progenitors along radial glial during brain development. We hypothesized that Cx43 could regulate the bidirectional migration of HSC/P in the BM stroma. Since Cx43 is expressed by mesenchymal cells, endothelial cells and hematopoietic stem cells and progenitors, we decided to analyze the Cx43 contribution in the control of HSC/P migration in cell-specific conditional knock-out mice. To achieve this objective, we have used mice that were selectively deficient for Cx43 in the osteoblast/stromal cells (Collagen 1a-Creflox/flox; O/S-Cx43-deficient), in endothelial cells (Tek-Creflox/flox; E-Cx43-deficient) or in hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). O/S-Cx43-deficient mice have been shown to be a model of osteoblast loss of function (Chung DJ et al., J. Cell. Sci., 2006) and E-Cx43-deficient mice have been shown to be a model of arterial hypotension induced by both increase nitric oxide and angiotensin levels (Liao Y et al, PNAS 2001). Analysis with reporter crossings with Rosa-loxP-Stop-LoxP-LacZ mice showed anatomical specificity of the Cre recombinase expression in different cell types of BM, and western-blot and RT-PCR expression indicated practical abolishment of the expression of Cx43 in each of the specific cell types. First, we analyzed whether there were changes in the levels of circulating progenitors in O/S-, E- or H-Cx43-deficient mice. While H-Cx43-deficient mice did not show any change in the levels of circulating HSC/P, E-Cx43-deficient mice showed a 3.5-fold and 4.7-fold, respectively, increase of circulating CFU-C and competitive repopulating units while maintaining normal repopulation ability of BM HSC. O/S-Cx43-deficient mice showed a 30% reduction in basal conditions which was more accentuated when administered G-CSF (50% reduction on day +6), compared with their WT counterparts. Interestingly, while osteoblast loss-of-function was induced in O/S Cx43-deficient mice, the intramarrow expression levels of CXCL12a/b and mesenchymal progenitor content (CFU-F) were increased (4- and 2-fold, respectively). In correlation with the increased levels of CXCL12, the distance to endosteum of transplanted CFSE+/lin-/c-kit+ BM cells into non-myeloablated O/S-Cx43-deficient mice was dramatically decreased (36.1±4.3 vs 23.2±2.1 mm, p<0.01), suggesting a major change in the cellular composition and chemokinesis within the hematopoietic microenvironment “in vivo”. Interestingly, the 16-hour homing of HSC/P transplanted into lethally irradiated O/S-Cx43KO recipient mice showed a ∼60% reduction and a significantly decreased survival in a limiting-dose transplantation radioprotection assay (50% survival in WT mice vs 0% survival in O/S Cx43-deficient recipients). The homing/engraftment defect of these mice correlated with a reversal of the increased levels of CXCL12 in irradiated BM and a 50% reduction of the migration of WT HSC/P through O/S-Cx43-deficient stroma in response to CXCL12. Altogether, these data indicate that intercellular communication through Cx43 shares distinct functions between the different cell components of the hematopoietic microenvironment, and mediates CXCL12-dependent and CXCL12-independent mechanisms in control of the BM homing and retention of HSC/P. Disclosures: No relevant conflicts of interest to declare.

Prolonged Expression of c-fos Suppresses Cell Cycle Entry of Dormant Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 816-825 ◽  
Author(s):  
Seiji Okada ◽  
Tetsuya Fukuda ◽  
Kunimasa Inada ◽  
Takeshi Tokuhisa
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Colony Formation ◽  
Cell Cycle Progression ◽  
Fetal Liver ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells ◽  
Deficient Mice

The proto-oncogene c-fos was transiently upregulated in primitive hematopoietic stem (Lin−Sca-1+) cells stimulated with stem cell factor, interleukin-3 (IL-3), and IL-6. To investigate a role of the c-fos in hematopoietic stem cells, we used bone marrow (BM) cells from transgenic mice carrying the c-fos gene under the control of the interferon-/β–inducible Mx-promoter (Mx–c-fos), and fetal liver cells from c-fos–deficient mice. Prolonged expression of the c-fos in Lin−Sca-1+ BM cells inhibited factor-dependent colony formation and hematopoiesis on a stromal cell layer by keeping them at G0/G1 phase of the cell cycle. These Lin−Sca-1+ BM cells on a stromal layer entered into the cell cycle whenever exogenous c-fos was downregulated. However, ectopic c-fos did not perturb colony formation by Lin−Sca-1+ BM cells after they entered the cell cycle. Furthermore, endogenous c-fos is not essential to cell cycle progression of hematopoietic stem cells because the factor-dependent and the stroma-dependent hematopoiesis by Lin−Sca-1+ fetal liver cells from c-fos–deficient mice was not impaired. These results suggest that the c-fos induced in primitive hematopoietic stem cells negatively controls cell cycle progression and maintains them in a dormant state.

Prolonged Expression of c-fos Suppresses Cell Cycle Entry of Dormant Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 816-825 ◽  
Author(s):  
Seiji Okada ◽  
Tetsuya Fukuda ◽  
Kunimasa Inada ◽  
Takeshi Tokuhisa
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Colony Formation ◽  
Cell Cycle Progression ◽  
Fetal Liver ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells ◽  
Deficient Mice

Abstract The proto-oncogene c-fos was transiently upregulated in primitive hematopoietic stem (Lin−Sca-1+) cells stimulated with stem cell factor, interleukin-3 (IL-3), and IL-6. To investigate a role of the c-fos in hematopoietic stem cells, we used bone marrow (BM) cells from transgenic mice carrying the c-fos gene under the control of the interferon-/β–inducible Mx-promoter (Mx–c-fos), and fetal liver cells from c-fos–deficient mice. Prolonged expression of the c-fos in Lin−Sca-1+ BM cells inhibited factor-dependent colony formation and hematopoiesis on a stromal cell layer by keeping them at G0/G1 phase of the cell cycle. These Lin−Sca-1+ BM cells on a stromal layer entered into the cell cycle whenever exogenous c-fos was downregulated. However, ectopic c-fos did not perturb colony formation by Lin−Sca-1+ BM cells after they entered the cell cycle. Furthermore, endogenous c-fos is not essential to cell cycle progression of hematopoietic stem cells because the factor-dependent and the stroma-dependent hematopoiesis by Lin−Sca-1+ fetal liver cells from c-fos–deficient mice was not impaired. These results suggest that the c-fos induced in primitive hematopoietic stem cells negatively controls cell cycle progression and maintains them in a dormant state.

scholarly journals Mitochondrial Fate of Regenerative Hematopoietic Stem Cells Is Sequentially Controlled By Two Specific Conformations of Connexin-43

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Abhishek K Singh ◽  
Ashley M Wellendorf ◽  
Breanna Bohan ◽  
Daniel Gonzalez-Nieto ◽  
Luis C Barrio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Connexin 43 ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Inhibitory Effect ◽  
Mitochondrial Fragmentation ◽  
Hematopoietic Stem

Restricted mitochondrial metabolism with low mitochondrial reactive oxygen species (ROS) and membrane potential are essential properties of repopulating hematopoietic stem cells (HSC). Upon regenerative stress, as found after chemotherapy and/or radiotherapy, HSC exit quiescence, proliferate and differentiate into mature blood cells. Understanding the mechanism controlling hematopoiesis regeneration upon replicative stress is expected to provide molecular targets for amelioration of chemotherapy induced toxicity on HSC. Recent evidence demonstrates that the coordinated regulation of mitochondrial dynamics and the clearance of damaged mitochondria are the critical determinants of HSC fate decisions. Upon myeloablative stress, hematopoietic connexin 43 (H-Cx43), a major component of the gap junctions (GJ) present in the cell, lysosome and mitochondrial membranes, preserves the survival and efficient blood formation of regenerating HSC and progenitors (HSPC) by the transfer of damaging excess ROS, preventing HSPC apoptosis and lethal hematology failure. The protective role of H-Cx43 depends on the regulation of cell-contact dependent mitochondrial transfer to BM mesenchymal stromal cells. Mitochondrial homeostasis is maintained by coordinated regulation of mitochondrial fission, fusion and lysosome dependent mitophagy. We hypothesized that hematopoietic Cx43 may exert a mitochondrial autonomous activity affecting the ability of HSC to regenerate. We created HSC mitochondrial reporter mice with hematopoietic deficiency of Cx43(H-Cx43D/D) and analyzed mitochondrial dynamics and fate in quiescent and dividing HSC. While quiescent Cx43D/D HSC function normally, Cx43 deficiency results in increased mitochondrial ROS and membrane depolarization in cycling HSC. Time lapsed imaging of photo-converted mitochondria indicate that mitochondria of Cx43D/D cycling HSC split into highly-motile, smaller fragments. Interestingly, the activating phosphorylation (Ser616) of the mitochondrial fission protein, Drp1 and its accumulation within mitochondria is higher in Cx43D/D dividing HSC. The recruitment of Drp1 to mitochondria is regulated by mitochondrial membrane adaptors Mff and Fis1. Expression of Fis1, but not Mff, is significantly increased in Cx43D/D cycling HSC. In contrast, the components of mitochondrial fusion machinery Mfn2 and active Drp1 (phospho-Drp1-Ser637) are significantly attenuated in dividing Cx43D/D HSC, suggesting that HSC Cx43 promotes mitochondrial fusion and stability, and inhibits mitochondrial fragmentation. Increased mitochondrial fission in dividing Cx43D/D HSC facilitates mitophagy as indicated by increased co-localization of mitochondria with the ubiquitin kinase Pink1 which simultaneously recruits the E3 ubiquitin ligase Parkin, autophagosome p62 and Lc3, and the lysosomal membrane protein Lamp2 on the surface of dysfunctional mitochondria. Additionally, increased phosphorylation of Ampk (Tyr172) and Ulk1 (Ser555) in mitochondria of cycling Cx43D/D HSC demonstrate that H-Cx43 is a negative regulator of Ampk dependent mitophagy in diving HSC. Inhibition of the Drp1 GTPase activity by expression of the dominant negative Drp1-K38A mutant prevents mitochondrial fragmentation, motility and mitophagy of dividing Cx43D/D HSC, confirming that the inhibitory effect on mitophagy of Cx43 depends on its role on mitochondrial fission. Expression of Cx43 structure-function mutants (cys-less mutant with impaired head-to-head hemichannel docking, but not hemichannel function; and C-terminus truncated D257 mutant with impaired signaling and intramolecular interactions needed for channel gating) in H-Cx43D/D HSC demonstrated that the negative regulatory role of Cx43 on mitochondrial fission requires functional Cx43 hemichannels while the constitutive inhibitory effect of H-Cx43 on mitophagy depends on the formation of complete functional GJ channels. Our results identify for first time the sequential role of two distinct conformations of mitochondrial H-Cx43 dependent channels on the control of mitochondrial fate: fission and mitophagy, in cycling HSC. This data provides novel targets for ex-vivo intervention to preserve HSC activity by transfer of genetically manipulated mitochondria. Figure Disclosures Cancelas: TerumoBCT: Consultancy, Research Funding; Cerus Corp: Research Funding; Hemanext Inc.: Consultancy, Research Funding; Velico LLC: Consultancy, Research Funding; Cytosorbents: Research Funding; Westat Inc: Consultancy, Research Funding; US DoD: Research Funding; NIH: Consultancy, Research Funding.

Genetic Deletion of Bcl-x Reveals a Potential Role in Governing Stem Cell Homeostasis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1349-1349
Author(s):  
Brenda J. Chyla ◽  
Jason G. Harb ◽  
Claudia S. Huettner
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Hematopoietic Stem ◽  
Final Maturation ◽  
Genetic Deletion ◽  
Deficient Mice

Abstract Germline deletion of bcl-x demonstrated the importance of this gene for hematopoiesis. Bcl-x deficient embryos failed to thrive due to massive apoptosis of immature hematopoietic and neural cells. Deletion of this gene in the adult has uncovered its requirement for the final maturation of erythroid cells, the transition of pre-B cells to the pro-B cell stage and recently, we showed an important role in the development of natural killer cells. While the role of bcl-x in hematopoietic stem cells (HSC) has yet to be addressed, a role for other members of anti-apoptotic bcl-2 family of proteins has been established. Overexpression of bcl-2 led to accumulation of HSC, whereas genetic deletion of mcl-1 resulted in ablation of HSC. In the present study, we determined by quantitative Real-Time PCR, that in addition to mcl-1, bcl-x is also highly expressed in hematopoietic stem cells, whereas the level of bcl-2 is threefold lower. Moreover, we found the expression of bcl-x to be down regulated with differentiation of HSC into progenitor populations (i.e. GMP, CMP and MEP). Therefore, we established a model to delete bcl-x in the stem cells of adult mice. We bred the Mx1-cre transgenic line with mice that carry the bcl-x gene flanked by loxP sites. Deletion of bcl-x occurred following administration of pIpC. A total of 10 bcl-x deficient mice and 7 age-matched control animals (Mxi-cre/wildtype bcl-x) were examined. Quantitative real-time PCR revealed a 55 – 95% reduction of bcl-x mRNA in Lin−, Sca-1+, c-kit+ cells 8 days post the first administration of pIpC. This population consists of phenotypically and functionally defined long-term HSC (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitors. We used FACS analysis to identify LT-HSC versus ST-HSC populations and to further assess the effects of bcl-x deletion. LT-HSC were defined by staining, for the following cell surface markers as Lin−, Sca-1+, c-kit+, Flt3−, CD34−; ST-HSC were characterized as Lin−, Sca-1+, c-kit+, Flt3−, CD34+. We found a 2 fold shift in the ratio of LT-HSC versus ST-HSC in bcl-x deficient mice compared to the ratio in control animals. This result raises the hypothesis that bcl-x regulates entry of HSC into the cell cycle. A notion that is supported by the observation that overexpression of bcl-x in fibroblasts delays transition from the Go/G1 to the S phase of the cell cycle. Future experiments will determine if the observed phenomenon is mediated through the anti-apoptotic function or cell cycle activity of the bcl-x protein.

scholarly journals Absence of Evidence Implicating Hematopoietic Stem Cells As Common Progenitors for DLBCL Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4107-4107
Author(s):  
Max Jan ◽  
Florian Scherer ◽  
David M. Kurtz ◽  
Aaron M Newman ◽  
Henning Stehr ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
B Cells ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Phylogenetic Trees ◽  
Somatic Mutations ◽  
Hematopoietic Stem ◽  
Tumor Biopsies ◽  
Myd88 L265p

Abstract Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs. Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs. Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs. Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

scholarly journals Single-cell RNA-seq reveals a concomitant delay in differentiation and cell cycle of aged hematopoietic stem cells

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Léonard Hérault ◽  
Mathilde Poplineau ◽  
Adrien Mazuel ◽  
Nadine Platet ◽  
Élisabeth Remy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Cycle Regulators ◽  
Potential Mechanism ◽  
Rna Seq ◽  
Hematopoietic Stem ◽  
Age Related ◽  
Undifferentiated State ◽  
Reference Map

Abstract Background Hematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging, heterogeneity of HSCs changes, contributing to the deterioration of the immune system. In this study, we revisited mouse HSC compartment and its transcriptional plasticity during aging at unicellular scale. Results Through the analysis of 15,000 young and aged transcriptomes, we identified 15 groups of HSCs revealing rare and new specific HSC abilities that change with age. The implantation of new trajectories complemented with the analysis of transcription factor activities pointed consecutive states of HSC differentiation that were delayed by aging and explained the bias in differentiation of older HSCs. Moreover, reassigning cell cycle phases for each HSC clearly highlighted an imbalance of the cell cycle regulators of very immature aged HSCs that may contribute to their accumulation in an undifferentiated state. Conclusions Our results establish a new reference map of HSC differentiation in young and aged mice and reveal a potential mechanism that delays the differentiation of aged HSCs and could promote the emergence of age-related hematologic diseases.

scholarly journals Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4640-4640
Author(s):  
Heng-Yi Liu ◽  
Nezia Rahman ◽  
Tzu-Ting Chiou ◽  
Satiro N. De Oliveira
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Lines ◽  
Immune Cells ◽  
Research Funding ◽  
Humanized Mice ◽  
Tumor Challenge ◽  
Hematopoietic Stem

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

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