scholarly journals Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirreCells

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Muti ur Rehman Khan ◽  
Ijaz Ali ◽  
Wei Jiao ◽  
Yun Wang ◽  
Saima Masood ◽  
...  
Keyword(s):  
Cell Line ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Critical Role ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem

Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34+cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF-βwith other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34+CD38−cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

scholarly journals The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells

Blood ◽  
2020 ◽  
Author(s):  
Lijian Shao ◽  
Adedamola Elujoba-Bridenstine ◽  
Katherine E Zink ◽  
Laura M Sanchez ◽  
Brian J Cox ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Direct Role ◽  
Hematopoietic Stem ◽  
Knockout Mouse Model ◽  
Immunodeficient Mice ◽  
Bm Niche

Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the g-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry (IMS) and found that endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1 knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared to wild-type littermates. Moreover, Gabbr1 null hematopoietic stem cells (HSCs) had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1 null HSPCs were less proliferative under steady-state conditions and upon stress. Colony assays demonstrated that almost all Gabbr1 null HSPCs were in a slow or non-cycling state. In vitro differentiation of Gabbr1 null HSPCs in co-cultures, produced fewer overall cell numbers with significant defects in differentiation and expansion of the B cell lineage. To determine if GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared to GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

scholarly journals Ex vivo expansion of human hematopoietic stem and progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6083-6090 ◽  
Author(s):  
Ann Dahlberg ◽  
Colleen Delaney ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Notch Signaling ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Growth Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

AbstractDespite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field.

Large Ex Vivo Expansion of Human Umbilical Cord Blood CD4+ and CD8+ T Cells

1999 ◽  
Vol 8 (2) ◽  
pp. 129-139 ◽  
Author(s):  
Danna Skea ◽  
Nan-Hua Chang ◽  
Robin Hedge ◽  
Barbara Dabek ◽  
Truman Wong ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Cd8 T Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord

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.

scholarly journals Ex Vivo Expansion of Human Umbilical Cord Blood-Derived T-Lymphocytes with Homologous Cord Blood Plasma

2005 ◽  
Vol 205 (2) ◽  
pp. 115-122 ◽  
Author(s):  
Yong-Man Kim ◽  
Min-Hyung Jung ◽  
Ha-Young Song ◽  
Hyun Ok Yang ◽  
Sung-Tae Lee ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Blood Plasma ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord

Characterization of ‘Early’ vs ‘Late’ Endothelial Progenitor Cells (EPCs) Which Are Derived from Human Umbilical Cord Blood (HCB) during Ex Vivo Expansion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1706-1706
Author(s):  
Eun-Sun Yoo ◽  
Jee-Young Ahn ◽  
Yun-Kyung Bae ◽  
Seung-Eun Lee ◽  
Sang min Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Tube Formation ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord ◽  
Vascular Injuries ◽  
Rt Pcr ◽  
Different Types

Abstract EPCs have been isolated from adult peripheral blood and bone marrow. Recently, several groups reported that two types (‘early’ & ‘late’) of EPC could be isolated from peripheral blood and bone marrow when pertinent cocktails of cytokines were used. Interestingly, early and late EPCs are different in terms of expression of surface markers, the abilities of tube formation in vitro and the capabilities of re-vascularization on hind limb ischemia models in mice. We found EPC formation during ex vivo expansion of HCB and one EPC could be found from 314 CD34+ cells from HCB based on limiting dilutional assay (ref. Stem Cells; 2003, Yoo et al). However, little is known about the characteristics of ‘early’ and ‘late’ EPCs that are derived from HCB. In this study, our aims are to isolate the ‘early’ and ‘late’ EPCs from HCB during ex-vivo HCB expansion period and to characterize the biologic properties between ‘early’ and ‘late’ EPCs. 1 x 108 mononuclear cells were plated on a 100mm culture dish coated with 50ug/ml of human fibronectin (Calbiochem) and cultured in EGM-2 BulletKit system (Clonetics). Endothelial cells were assessed by colony counts, flow cytometry, proliferation assay, RT-PCR and in vitro tube formation in Matrigel plate. Migration of EPCs were also measured by in vitro transmigration assay in the presence of VEGF and SDF-1. In results, early spindle-shaped cells (‘early’ EPCs) which were grown at first week of culture were positive for CD31, CD14 and CXCR-4. Cobblestone shaped cells (‘late’ EPC) were in peak growth at second and third weeks of culture and were also positive using above antibodies except CD14. Early EPCs had not expressed mRNA of KDR, vWF and VE-Cadherin by RT-PCR. However, late EPCs expressed high level of mRNA of those endothelial marker genes. Both early and late EPCs expressed mRNA of eNOS. Late EPC produced more nitric oxide and formed more capillary tubes than those of early spindle-shaped cells. Early EPCs were readily migrated by VEGF and SDF-1 compared with those of late EPCs. In conclusions, we have found two different types of EPCs with different biologic properties during HCB ex vivo expansion. These findings may have potential clinical applications for “cell therapy” on vascular injuries (ie, hindlimb ischemia and myocardial infacrtion). Murine models for vascular injuries are being established to test the efficacy of different types of EPCs from HCB in our Lab.

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