scholarly journals Engineering human megakaryocytic microparticles for targeted delivery of nucleic acids to hematopoietic stem and progenitor cells

2018 ◽  
Vol 4 (11) ◽  
pp. eaau6762 ◽  
Author(s):  
Chen-Yuan Kao ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Progenitor Cells ◽  
Targeted Delivery ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Patient Specific ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Hematopoietic stem and progenitor cells (HSPCs) are important target cells for gene therapy applications. Current genetic modifications of HSPCs rely on viral vectors in vivo or electroporation ex vivo. Here, we developed a nonviral system based on megakaryocytic microparticles (MPs) for targeted delivery of plasmid DNA (pDNA) and small RNAs to HSPCs. We have previously shown that megakaryocytic MPs, the most abundant MPs in blood circulation, target specifically and deliver cargo to HSPCs both in vitro and in vivo. With an optimized electroporation protocol, an average of 4200 plasmid copies per MP were loaded into MP, thus enabling effective delivery of green fluorescent protein (GFP)–encoding pDNA to HSPCs and HSPC nuclei, with up to 81% nuclei containing pDNA. Effective functional small interfering RNA (siRNA) and microRNA (miRNA) delivery were also demonstrated. As patient-specific or generic megakaryocytic MPs can be readily generated and stored frozen, our data suggest that this system has great potential for therapeutic applications targeting HSPCs.

scholarly journals Extracellular vesicles from human iPSCs enhance reconstitution capacity of cord blood-derived hematopoietic stem and progenitor cells

Leukemia ◽  
2021 ◽  
Author(s):  
Elżbieta Karnas ◽  
Małgorzata Sekuła-Stryjewska ◽  
Katarzyna Kmiotek-Wasylewska ◽  
Sylwia Bobis-Wozowicz ◽  
Damian Ryszawy ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Extracellular Vesicles ◽  
Functional Properties ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Human Ipscs ◽  
Stem And Progenitor Cells

AbstractCord blood (CB) represents a source of hematopoietic stem and progenitor cells (CB-HSPCs) for bone marrow (BM) reconstitution, but clinical CB application is limited in adult patients due to the insufficient number of CB-HSCPCs and the lack of effective ex vivo approaches to increase CB-HSPC functionality. Since human-induced pluripotent stem cells (hiPSCs) have been indicated as donor cells for bioactive extracellular vesicles (EVs) modulating properties of other cells, we are the first to employ hiPSC-derived EVs (hiPSC-EVs) to enhance the hematopoietic potential of CB-derived CD45dimLin-CD34+ cell fraction enriched in CB-HSPCs. We demonstrated that hiPSC-EVs improved functional properties of CB-HSPCs critical for their hematopoietic capacity including metabolic, hematopoietic and clonogenic potential as well as survival, chemotactic response to stromal cell-derived factor 1 and adhesion to the model components of hematopoietic niche in vitro. Moreover, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. This phenomenon might be related to activation of signaling pathways in CB-HSPCs following hiPSC-EV treatment, as shown on both gene expression and the protein kinases activity levels. In conclusion, hiPSC-EVs might be used as ex vivo modulators of CB-HSPCs capacity to enhance their functional properties and augment future practical applications of CB-derived cells in BM reconstitution.

Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1557-1566 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Michael J. Bass ◽  
Gary Van Zant ◽  
Barry Grimes
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hematopoietic Reconstitution ◽  
Dramatic Decline ◽  
Stem And Progenitor Cells ◽  
Clonogenic Cells

Abstract Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1387-1387
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 83-83
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Ann Atzberger ◽  
Dengli Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

scholarly journals In vitro and in vivo evaluation of cord blood hematopoietic stem and progenitor cells amplified with glycosaminoglycan mimetic

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Lionel Faivre ◽  
Véronique Parietti ◽  
Fernando Siñeriz ◽  
Sandrine Chantepie ◽  
Marie Gilbert-Sirieix ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
In Vivo Evaluation ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

scholarly journals Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells

2017 ◽  
Vol 216 (7) ◽  
pp. 2217-2230 ◽  
Author(s):  
Gregoire Stik ◽  
Simon Crequit ◽  
Laurence Petit ◽  
Jennifer Durant ◽  
Pierre Charbord ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Critical Role ◽  
Molecular Signature ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver–derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

Enforced expression of cyclin D2 enhances the proliferative potential of myeloid progenitors, accelerates in vivo myeloid reconstitution, and promotes rescue of mice from lethal myeloablation

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 986-992 ◽  
Author(s):  
Yutaka Sasaki ◽  
Christina T. Jensen ◽  
Stefan Karlsson ◽  
Sten Eirik W. Jacobsen
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Cyclin D2 ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Myeloid Progenitors

AbstractSevere and prolonged cytopenias represent a considerable problem in clinical stem cell transplantations. Cytokine-induced ex vivo expansion of hematopoietic stem and progenitor cells has been intensively explored as a means of accelerating hematopoietic recovery following transplantation but have so far had limited success. Herein, overexpression of D-type cyclins, promoting G0/G1 to S transition, was investigated as an alternative approach to accelerate myeloid reconstitution following stem cell transplantation. With the use of retroviral-mediated gene transfer, cyclin D2 was overexpressed in murine bone marrow progenitor cells, which at limited doses showed enhanced ability to rescue lethally ablated recipients. Competitive repopulation studies demonstrated that overexpression of cyclin D2 accelerated myeloid reconstitution following transplantation, and, in agreement with this, cyclin D2–transduced myeloid progenitors showed an enhanced proliferative response to cytokines in vitro. Furthermore, cyclin D2–overexpressing myeloid progenitors and their progeny were sustained for longer periods in culture, resulting in enhanced and prolonged granulocyte production in vitro. Thus, overexpression of cyclin D2 confers myeloid progenitors with an enhanced proliferative and granulocyte potential, facilitating rapid myeloid engraftment and rescue of lethally ablated recipients.

scholarly journals IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors

2020 ◽  
Vol 217 (9) ◽  
Author(s):  
James W. Swann ◽  
Lada A. Koneva ◽  
Daniel Regan-Komito ◽  
Stephen N. Sansom ◽  
Fiona Powrie ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Chronic Inflammation ◽  
Inflammatory Disease ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

An important comorbidity of chronic inflammation is anemia, which may be related to dysregulated activity of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Among HSPCs, we found that the receptor for IL-33, ST2, is expressed preferentially and highly on erythroid progenitors. Induction of inflammatory spondyloarthritis in mice increased IL-33 in BM plasma, and IL-33 was required for inflammation-dependent suppression of erythropoiesis in BM. Conversely, administration of IL-33 in healthy mice suppressed erythropoiesis, decreased hemoglobin expression, and caused anemia. Using purified erythroid progenitors in vitro, we show that IL-33 directly inhibited terminal maturation. This effect was dependent on NF-κB activation and associated with altered signaling events downstream of the erythropoietin receptor. Accordingly, IL-33 also suppressed erythropoietin-accelerated erythropoiesis in vivo. These results reveal a role for IL-33 in pathogenesis of anemia during inflammatory disease and define a new target for its treatment.

scholarly journals Liver Sinusoidal Endothelial Cells Promote the Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells

2019 ◽  
Vol 20 (8) ◽  
pp. 1985 ◽  
Author(s):  
Huilin Li ◽  
Haiyun Pei ◽  
Xiaoyan Xie ◽  
Sihan Wang ◽  
Yali Jia ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Liver Sinusoidal Endothelial Cells ◽  
Sinusoidal Endothelial Cells ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34+ hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34+CD38− and CD34+ CD38−CD90+ HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34+ hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34+ hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.

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