Signal transducer and activator of transcription 5a (STAT5a) is required for eosinophil differentiation of human cord blood–derived CD34+ cells

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 134-142 ◽  
Author(s):  
Miranda Buitenhuis ◽  
Belinda Baltus ◽  
Jan-Willem J. Lammers ◽  
Paul J. Coffer ◽  
Leo Koenderman
Keyword(s):  
Cord Blood ◽  
Target Genes ◽  
Ex Vivo ◽  
Critical Role ◽  
Ectopic Expression ◽  
Hematopoietic Cells ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Human Cord Blood ◽  
Cd34 Cells

Abstract Signal transducers and activators of transcription (STATs) have been reported to play a critical role in the differentiation of several myeloid cell lines, although the importance of STATs in the differentiation of primary human hematopoietic cells remains to be established. Terminal eosinophil differentiation is induced by interleukin-5 (IL-5), which has also been demonstrated to activate STAT5. We have investigated whether STAT5 plays a critical role during eosinophil differentiation using umbilical cord blood–derived CD34+ cells. In this ex vivo system, STAT5 expression and activation are high early during differentiation, and STAT5 protein expression is down-regulated during the final stages of eosinophil differentiation. Retroviral transductions were performed to ectopically express wild-type and dominant-negative STAT5a (STAT5aΔ750) in CD34+ cells. Transduction of cells with STAT5a resulted in enhanced proliferation compared with cells transduced with empty vector alone. Interestingly, ectopic expression of STAT5a also resulted in accelerated differentiation. In contrast, ectopic expression of STAT5aΔ750 resulted in a block in differentiation, whereas proliferation was also severely inhibited. Similar results were obtained with dominant-negative STAT5b. Forced expression of STAT5a enhanced expression of the STAT5 target genes Bcl-2 andp21WAF/Cip1, suggesting they may be important in STAT5a-mediated eosinophil differentiation. These results demonstrate that STAT5 plays a critical role in eosinophil differentiation of primary human hematopoietic cells.

scholarly journals Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression

2001 ◽  
Vol 16 (1) ◽  
pp. 20 ◽  
Author(s):  
Ju Young Seoh ◽  
Hae Young Park ◽  
Wha Soon Chung ◽  
Seung Cheol Kim ◽  
Myong Joon Hahn ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Antigen Expression ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Late Antigen ◽  
Cord Blood Cd34 ◽  
Cell Cycling

Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2888-2888
Author(s):  
Ana Frias ◽  
Christopher D. Porada ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Cell Number ◽  
Human Cord Blood ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1329-1329
Author(s):  
Aleksandra Rizo ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
Jan Jacob Schuringa
Keyword(s):  
Cord Blood ◽  
Cell Expansion ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

scholarly journals Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin

2001 ◽  
Vol 113 (2) ◽  
pp. 470-478 ◽  
Author(s):  
Kyung-Ha Ryu ◽  
Susan Chun ◽  
Steve Carbonierre ◽  
Seock-Ah Im ◽  
Hyung-Lae Kim ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Megakaryocytic Differentiation ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Chromosome analysis after ex vivo expansion of CD34+ cells from human cord blood

2001 ◽  
Vol 125 (2) ◽  
pp. 161-162 ◽  
Author(s):  
Yoshio Katayama ◽  
Kanji Miyamoto ◽  
Katsuto Takenaka ◽  
Kenji Imajyo ◽  
Katsuji Shinagawa ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Chromosome Analysis ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Cd34 Cells

scholarly journals Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4332-4332
Author(s):  
Xinxin Huang ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Lentiviral Vectors ◽  
Ex Vivo Expansion ◽  
Transcriptional Factor ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

In Vivo Expansion of Transduced Human Erythroid Cells Using an Mpl-Based Cell Growth Switch.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2100-2100
Author(s):  
Yasuo Nagasawa ◽  
Brent Wood ◽  
Cynthia L. Nourigat ◽  
Thalia Papayannopoulou ◽  
C. Anthony Blau
Keyword(s):  
Cell Growth ◽  
Cord Blood ◽  
Genetically Modified ◽  
Hematopoietic Cells ◽  
Erythroid Cells ◽  
Post Transplantation ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Cell growth switches are engineered signaling molecules that can trigger cell growth in response to artificial ligands such as chemical inducers of dimerization (CIDs). We have previously shown that a cell growth switch comprised of the intracellular portion of the thrombopoietin receptor, Mpl, allows for the CID-dependent, in vivo expansion of genetically modified primary hematopoietic cells in mouse and dog models. Here we report the application of this approach to the in vivo expansion of genetically modified primary human hematopoietic cells using an immune deficient mouse model. A lentivirus vector encoding a CID-activatible deriviative of Mpl (F36VMpl) and a green fluorescent protein (GFP) reporter was used to transduce human cord blood CD34+ cells. Transduced human cord blood CD34+ cells expanded 347–495 fold in cultures containing no added growth factors and 100 nM of AP20187. We proceeded to test CID-responsiveness following transplantation into NOD-SCID-beta 2 microglobulin null mice. Since significant human red cell engraftment persists for only a few weeks post transplantation in this model, mice were evaluated at 3 weeks post transplantation, following a 2 week course of treatment with either CID (AP20187 10 mg/kg/day) or control vehicle alone (without CID). In 2 experiments totalling 42 mice, CID-administration resulted in a significant rise in GFP positive human cells, with the predominant response occuring among human erythroid cells (exp.1: 4.0 fold, P = 0.0003, exp.2: 12.7 fold, P = 0.038). An increase in transduced human erythroid progenitor cells was observed in the spleens (but not the femurs) of CID treated mice. Effects on other hematopoietic lineages were minor and variable. The effect of CID treatment was no longer evident five weeks after the last dose. The restriction of CID-induced cell growth to the erythroid lineage may make this approach well suited for gene therapy applications in sickle cell anemia and beta thalassemia.

The tetraspanin CD9 regulates migration, adhesion, and homing of human cord blood CD34+ hematopoietic stem and progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (6) ◽  
pp. 1840-1850 ◽  
Author(s):  
Kam Tong Leung ◽  
Kathy Yuen Yee Chan ◽  
Pak Cheung Ng ◽  
Tze Kin Lau ◽  
Wui Man Chiu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Critical Role ◽  
Janus Kinase ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Short Term Exposure ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract The stromal cell–derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing and engraftment of hematopoietic stem/progenitor cells (HSCs) during bone marrow transplantation. To investigate the transcriptional regulation provided by this axis, we performed the first differential transcriptome profiling of human cord blood CD34+ cells in response to short-term exposure to SDF-1 and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family of proteins, was expressed in CD34+CD38−/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously down-regulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expression was modulated via CXCR4, G-protein, protein kinase C, phospholipase C, extracellular signal-regulated kinase, and Janus kinase 2 signals. Pretreatment of CD34+ cells with the anti-CD9 monoclonal antibody ALB6 significantly inhibited SDF-1–mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells was enhanced. Pretreatment of CD34+ cells with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice. Sorted CD34+CD9− cells displayed lower bone marrow homing capacity compared with that of total CD34+ cells. CD9 expression on homed CD34+ cells was significantly up-regulated in vivo. Our results indicate that CD9 might possess specific functions in HSC homing.

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