Lack of Self-Renewal Capacity in Fancc −/− Stem Cells After Ex Vivo Expansion

Stem Cells ◽  
2005 ◽  
Vol 23 (8) ◽  
pp. 1135-1141 ◽  
Author(s):  
Ouassila Habi ◽  
Marie-Chantal Delisle ◽  
Nancy Messier ◽  
Madeleine Carreau
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal

scholarly journals Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells

2015 ◽  
Vol 5 (4) ◽  
pp. 621-632 ◽  
Author(s):  
Gregory W. Charville ◽  
Tom H. Cheung ◽  
Bryan Yoo ◽  
Pauline J. Santos ◽  
Gordon K. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Muscle ◽  
Self Renewal ◽  
Muscle Stem Cells

Direct Evidence for Ex Vivo Expansion of Human Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 798-798
Author(s):  
Kiyoshi Ando ◽  
Takashi Yahata ◽  
Tadayuki Sato ◽  
Hiroko Miyatake ◽  
Hideyuki Matsuzawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Scid Mouse ◽  
Direct Evidence ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Ex vivo expansion of hematopoietic stem cells (HSC) is a major challenge for clinical and experimental transplantation protocols. However, no significant clinical benefit has been demonstrated to date. Clonal kinetics of ex vivo-expanded HSCs is one of the basic transplantation biology questions to be addressed before we can optimize ex vivo expansion approaches. To characterize human HSC, xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSC, LDA is a statistical method and does not directly establish that a single HSC has self renewed in vitro. By using lentiviral gene-marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. To detect multiplied clones, 5x104 gene-marked CD34+ cells were cultured for 4 days in our ex vivo expansion culture system (Exp Hematol, 27:904–915, 1999), and then divided into 10 lots, each of which was transplanted directly into the bone marrow of a NOD/SCID mouse. We used linear amplification-mediated (LAM)-PCR to detect unique genomic-proviral junctions as clonal markers. Detection of the same clones in different mice would provide direct evidence of ex vivo multiplication of a SRC clone. We identified 20 clone-specific genomic-proviral junction sequences by LAM-PCR on 10 mice. Although 14 clones were detected in only one mouse, six clones were detected in more than 2 mice. In the next experiment, purified CD19+EGFP+ and CD33+EGFP+ cells from each mouse were analyzed for each clone to detect multi-lineage differentiation of amplified SRCs. We identified 15 clonal markers from 6 mice. While 12 clones were present in only one mouse, 3 clones were present in 2 independent mice and reconstituted both CD19+and CD33+cells. Finally, we designed a secondary transplantation experiment to confirm the self-renewal ability of each clone. We identified 39 clonal markers from 10 primary and 10 secondary transplanted mice, 11 of which were detected in multiple mice with secondary transplantable ability. Together, of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than two mice after serum-free and stroma-dependent culture. Some of them were secondary transplantable. Furthermore, we identified new class of stem cells based not on repopulation, or cell surface markers, but on response to cytokine stimulation in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Study of the Mechanisms by Which Angptl5 and IGFBP2 Support Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2308-2308
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract We previously showed that angiopoietin-like protein 5 (Angptl5) and IGF Binding Protein 2 (IGFBP2) support dramatic ex vivo expansion of human hematopoietic stem cells (HSCs). To understand the mechanisms of their action, here we studied the effects of Angptl5 and IGFBP2 on the surface phenotype, signaling activation, self-renewal, apoptosis, differentiation, and homing of human cord blood CD34+ cells. Using immunofluorescence staining, we showed that Angptl5 and IGFBP2 activate certain signaling pathways such as MAPK and Stat5 in human cord blood CD34+ cells. IGFBP2 and Angptl5 increased the expression of transcription factors HoxB4, Bmi-1, EZH2, and survivin, measured by intracellular staining flow cytometry analysis and real-time RT-PCR. IGFBP2 and Angptl5 also inhibit expression of certain transcription factors important for differentiation of myeloid, erythroid, and lymphoid lineages. To test whether IGFBP2 and Angptl5 affect the homing of HSCs, we cultured human cord blood CD34+ cells in serum-free medium supplemented with SCF, TPO, Flt3-L, IGFBP2 or Angptl5, and transplanted them into sublethally irradiated NOD/SCID mice intraveneously or intrafemorally. Both IGFBP2 and Angptl5 support ex vivo expansion of SRCs in intrafemorally injected mice, suggesting the expansion-stimulating effects elicited by both factors are not caused by modulation of HSC homing. Interestingly, when we used intrafemoral injection, we found that Angptl5 treated HSCs have enhanced engraftment in non-injected bone marrow. This suggests Angptl5 treated HSCs further facilitate the mobilization of HSCs in vivo. We conclude that IGFBP2 and Angptl5 support self-renewal and inhibit differentiation of human cord blood HSCs. Our data also suggest that a combination of expression of transcription factors important for self-renewal, survival, and differentiation of HSCs can be used as a “stemness index” that predicts the activity of cultured human HSCs.

Direct evidence for ex vivo expansion of human hematopoietic stem cells

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3371-3377 ◽  
Author(s):  
Kiyoshi Ando ◽  
Takashi Yahata ◽  
Tadayuki Sato ◽  
Hiroko Miyatake ◽  
Hideyuki Matsuzawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Direct Evidence ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Asymmetric Divisions

AbstractTo characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Inhibition of p38 MAPK Promotes Hematopoietic Stem Cells Self-Renewal and Ex Vivo Expansion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 34-34
Author(s):  
Yong Wang ◽  
Joshua Kellner ◽  
Daohong Zhou
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
P38 Mapk ◽  
Cellular Senescence ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 34 Activation of the p38 mitogen-activated protein kinase (p38 MAPK) is implicated in the inhibitory effects of TNF-α, TGF-β, interferons and reactive oxygen species (ROS) on hematopoiesis and self-renewal of hematopoietic stem cells (HSCs). Clinically, overactivated p38 MAPK contributes to the pathogenesis of myelodysplastic syndromes (MDS) and Fanconi anemia. Inhibition of p38 MAPK with pharmacological agents improves hematopoietic progenitors' function in MDS. However, it has yet to be determined if p38 MAPK plays a role in regulation of normal HSC self-renewal and whether inhibition of p38 MAPK can improve HSC ex vivo expansion. In the present study, we found that sorted mouse bone marrow HSCs (Lin− Sca1+ c-kit+ cells or LSK+ cells) exhibited specific activation of p38 MAPK after seven days culture in serum-free medium supplemented with stem cell growth factors (SCF, Tpo and Flt3 ligand). The activation of p38 MAPK was associated with rapid differentiation of HSCs and induction of cellular senescence. Addition of SB203580 (SB, a specific p38 MAPK inhibitor) to the culture abrogated the activation of p38 MAPK, inhibited the induction of cellular senescence and increased the expression of several HSC self-renewing genes (such as CXCR4, HoxB4 and Gfi1). Moreover, HSCs cultured with SB resulted in a significantly greater HSC expansion than HSCs cultured without SB as assessed by flow cytometry and cobblestone area-forming cell (CAFC) assay. Finally, competitive repopulation assays revealed that HSCs expanded with SB produced a dramatic increase in donor-derived engraftments after transplantation to irradiated recipients. These findings suggest that p38 MAPK plays an important role in the regulation of HSC self-renewal and its inhibitors (e.g. SB203580) may be clinically useful in the ex vivo expansion of HSCs. Disclosures: No relevant conflicts of interest to declare.

Pyrimido-Indole Derivatives Are Novel Agonists of Human Cord Blood Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 650-650
Author(s):  
Iman Fares ◽  
Jalila Chagaroui ◽  
Yves Gareau ◽  
Stéphane Gingras ◽  
Nadine Mayotte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Fed Batch ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The widespread use of cord blood (CB) unit in transplantation is limited with low number of long-term hematopoietic stem cells (LT-HSCs) and progenitors. Several approaches have been developed to expand HSC ex vivo such as automated and continuous medium delivery (fed-batch), notch delta ligand and SR1 (antagonist of aryl hydrocarbon receptor (AhR)). Concurrent with these studies, we hypothesized that small molecule with potent LT-HSC stimulating activity might be identified and potentiated in fed-batch culture system. Accordingly, we tested a library of more than 5000 small molecules for their in vitro expansion of CD34+CD45RA- cells. Most of the identified hits, except one (UM729) synthesized in our institute, suppress AhR pathway. Structure activity relationship was performed on UM729 to generate a more potent analog named UM171. This optimized molecule was 10-20 times more potent with an effective concentration of 15-20 nM when tested for its ability to expand CD34+CD45RA- cells. When compared to SR1, UM171 delivered in a fed-batch system for 12 and 16 days showed a better expansion of HSC phenotypes and lower apoptotic cell number compared to SR1 or DMSO controls. Also, UM171-expaned cultures showed higher number in multipotent progenitors (CFU-GEMM) and long term initiating cells (LTC-IC) compared to DMSO controls. Further studies showed the UM171 did not affect division rate, and its effect in expanding HSC phenotype was reversible. When combined with SR1, UM171 showed a better suppression of differentiation and led to a higher CFU-GEMM expansion compared to the single treatment of the compounds or DMOS controls. These observations suggest that UM171+SR1 cooperate to enhance ex vivo expansion of progenitor cells and suppress differentiation. To determine the in vivo activity of the expanded CD34+ CB cells, we transplanted fresh (un-manipulated) and 12-day cultured cells in NSG mice and monitored the human hematopoietic reconstitution after 20 and 30 weeks post-transplantation. Frequencies of day0 equivalent LT-HSCs were 13-fold higher in UM171 expanded cultures compared to fresh or fed-batch cultures supplemented with DMSO or SR1. Secondary experiments indicated that UM171 ex vivo treatment did not appear to affect the capability of LT-HSC to expand in primary recipients and hence similarly reconstituted secondary animals for at least 18 more weeks. This suggests that UM171 expands LT-HSC ex vivo without losing their engraftment potential. To further investigate UM171 mechanism of action, RNA- Seq expression profiling was performed. Unlike SR1 or DMSO controls, UM171 treatment was accompanied by a marked suppression of transcripts associated with erythroid and megakaryocytic differentiation and up-regulation of membrane protein transcripts such as EPCR and TEMEM 183a. In summery, UM171 is the first molecule identified so far that enables a robust ex vivo expansion of human CD34+ CB cells that sustain their in vivo activity independent of AhR suppression. Conversely, AhR suppression was limited to expand cells with less durable self-renewal potential. This study could enhance the use of small yet well HLA-matched CB units to become a prioritized source for stem cells transplantation. Disclosures No relevant conflicts of interest to declare.

Alternative Xeno-Free Biomaterials Derived from Human Umbilical Cord for the Self-Renewal Ex-vivo Expansion of Mesenchymal Stem Cells

2013 ◽  
Vol 22 (22) ◽  
pp. 3025-3038 ◽  
Author(s):  
Sun-Mi Kim ◽  
Sung-Hwan Moon ◽  
Youngjun Lee ◽  
Gi Jin Kim ◽  
Hyung-Min Chung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Cord ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
The Self ◽  
Human Umbilical Cord ◽  
Self Renewal

Ex Vivo Expansion of Stem Cells: Defining Optimum Conditions Using Various Cytokines

2006 ◽  
Vol 12 (2) ◽  
pp. 86-93 ◽  
Author(s):  
Azza Mohamed ◽  
Azza Ibrahim ◽  
Manal El-Masry ◽  
Iman Mansour ◽  
Mervat Khroshied ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Optimum Conditions

scholarly journals Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells

2021 ◽  
Author(s):  
Valentina Orticelli ◽  
Andrea Papait ◽  
Elsa Vertua ◽  
Patrizia Bonassi Signoroni ◽  
Pietro Romele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem
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