Selected Members of the Angiopoietin-Like Family of Human Proteins Enhance Survival and Replating Capacity of Immature Human Hematopoietic Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3367-3367
Author(s):  
Hal E. Broxmeyer ◽  
Edward F. Srour ◽  
Scott Cooper ◽  
Carrie T. Wallace ◽  
Giao Hangoc ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Gm Csf ◽  
Cd34 Cells

Abstract Angiopoietin-like (ANGPTL) molecules are a family of secreted proteins which have characteristic structures of angiopoietins. This includes a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils (CC), a short linker peptide, and a globular fibrinogen-like domain (FLD). Zhang et. al. (Nat. Med., 12(2):240–245, 2006) reported that human ANGPTL-2, 3, 3CC, 5 and 7, but not ANGPTL4, enhanced ex-vivo expansion of highly enriched mouse bone marrow (BM) long term competitive repopulating hematopoietic stem cells in serum-free culture with SCF, TPO, IGF-2, and FGF-1. To the present, there have not been publications describing effects of human ANGPTL molecules on hematopoietic progenitor cells (HPC) or on human hematopoietic cells. Thus, we evaluated purified recombinant human ANGPTL-2CC, 3, 3CC, 3FLD, 4, 4CC, 5, 6 and 7 (AdipoGen, Inc, Seoul, Korea) for effects on proliferation and survival of HPC from human cord blood (CB). No endotoxin was detected in the ANGPTL molecule preparations (<0.1 EU/ug endotoxin per LAL method). None of the ANGPTL molecules at up to 500ng/ml stimulated HPC colony formation by themselves, or enhanced or inhibited HPC colony formation of low density (LD) or CD34+ human cord blood (CB) cells stimulated by GM-CSF, GM-CSF plus SCF, Epo plus SCF, or the combination of Epo, SCF, IL-3 and GM-CSF. However, ANGPTL-2CC, 3, and 3CC at 200 and 100, but not 10ng/ml significantly enhanced the survival of human LD and CD34+ HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors (Epo, SCF, IL-3, GM-CSF). Survival is a measure of anti-apoptosis for the hematopoietic progenitor cells in this context. The other ANGPTL molecules were not active at up to 500ng/ml. The survival enhancing effects of ANGPTL-3 was neutralized by purified rabbit anti-ANGPTL-3 IgG, but not by anti-ANGPTL-4, -6, or -7. Replating of HPC colonies offers an estimate of the self-renewal capabilities of HPC. We found that ANGPTL-3, but not -4, -6, or -7 enhanced the replating capacity of single CFU-GEMM colonies by greater than 2 fold. Thus far, we have not detected significant effects of the ANGPTL molecules on ex-vivo expansion of human CB CD34+ cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3, after assessing output of HPC, % and numbers of CD34+ cells, or cell cycle status of produced cells. In summary, we have implicated a few members of the ANGPTL family of proteins in functional effects on human HPC survival and replating/”self-renewal” activity, effects requiring the CC domain of the ANGPTL molecules. This information may be of relevance to regulation of HPC, and of use for protocols to use these cells for transplantation.

scholarly journals Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2059-2068 ◽  
Author(s):  
CM Traycoff ◽  
ST Kosak ◽  
S Grigsby ◽  
EF Srour
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Cell Tracking ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cells In Culture ◽  
Cd34 Cells ◽  
Limiting Dilution

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2376-2385 ◽  
Author(s):  
C Caux ◽  
B Vanbervliet ◽  
C Massacrier ◽  
I Durand ◽  
J Banchereau
Keyword(s):  
Tumor Necrosis Factor ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Langerhans Cells ◽  
Tumor Necrosis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Cord Blood Cd34 ◽  
Necrosis Factor

We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM- CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM- CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells.

scholarly journals A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1446-1454 ◽  
Author(s):  
N Katayama ◽  
M Nishikawa ◽  
F Komada ◽  
N Minami ◽  
S Shirakawa
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Hematopoietic Progenitor Cells ◽  
Dependent Manner ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Gm Csf ◽  
Erythroid Progenitor Cells ◽  
Dependent Inhibition ◽  
Dose Dependent

Abstract A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

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.

DPP4 (CD26) Truncation Of GM-CSF and IL-3 Alters Their Signaling and Functional Activity In Normal and Leukemic Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1206-1206 ◽  
Author(s):  
Heather A. O'Leary ◽  
Charlie Mantel ◽  
Xianyin Lai ◽  
Scott Cooper ◽  
Giao Hangoc ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Receptor Binding ◽  
Functional Activity ◽  
Colony Formation ◽  
Full Length ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Penultimate Proline

Abstract DPP4 (CD26) is a dipeptidyl peptidase that functions by enzymatically cleaving the penultimate proline, alanine or select other amino acids such as serine of proteins, resulting in functional alterations of the protein. We recently published that many cytokines, chemokines and growth factors have putative DPP4 truncations sites and that DPP4 specifically was able to truncate some colony stimulating factors such as GM-CSF and IL-3 with resultant blunting of their activity. However, the mechanism of action of the truncated factors is still unknown and requires further investigation. The expression, and activity, of DPP4 is relevant in normal and malignant hematopoiesis as we have data showing that CD34+ umbilical cord blood cells (UCB) as well as Acute Myelogenous Leukemia (AML) patient samples express active DPP4. Further, specific inhibition of DPP4 increases homing and engraftment of both human UCB and mouse bone marrow cells after transplantation in mice indicating the therapeutic potential of DPP4 activity altering compounds. Due to its potential importance in disease states, and their subsequent treatment, it is relevant to study how the activity of DPP4 alters the functions of the molecules it cleaves, and subsequently their interactions with each other. DPP4 can cleave the penultimate proline of GM-CSF and IL-3 resulting in truncated forms which have blunted colony stimulating factor activity for hematopoietic progenitor cells (HPC). Since GM-CSF and IL-3 receptors share a common receptor beta chain, we investigated if DPP4 truncation of GM-CSF (TGM) or IL-3 (T3) could inhibit the receptor binding and functional activity of the full length (FL) alternate compound (i.e TGM inhibition of FL3 activity or T3 inhibition of FLGM activity) in the factor dependent TF-1 cell line, UCB cells and in in vivo mouse studies. We determined using TF-1 and UCB that both T3 and TGM bound to the receptors with higher affinity than their FL forms and could blunt the receptor binding of the FLGM and FL3. Additionally, TGM and T3 decreased colony formation induced by either FLGM or FL3 in both TF-1, UCB, and primary AML patient cell samples. Strikingly, this inhibition of colony formation did not require a 1:1 ratio of the full length to truncated forms of these cytokines. Rather, approximately 4-10 fold less truncated molecules could be used to efficiently inhibit the colony formation activity of the full length form, even across molecules. In vivo injection of FL, T, or a mixture of FL/T or T/T factors into DPP4 activity knockout mice followed by colony assays showed the TGM and T3 suppresed the effect of FLGM or FL3 on progenitor cell numbers per femur and diminished cycling of hematopoietic progenitor cells as detected by high specificity tritiated thymidine kill assay. Proteomic analysis of the effects of full length and truncated factors (FLGM, FL3, TGM, T3) were performed with TF-1 cells where we detected differential protein regulation by the full length vs truncated factors. After 24 hour treatment with 10ng/ml of FLGM or TGM, TF-1 cells displayed statistically significant (p < .05) differences in 26 proteins of which 17 were higher in the FL vs the T, and 9 higher in the T vs FL treated groups. These proteins included, but were not limited to, cell cycle proteins such as CDK6, HDAC6, as well as signal transduction proteins and redox control proteins such as STAM1 and Glutaredoxin. Additionally, alterations in protein phosphphorylation were detected for TF-1 cells treated for 15 or 30 min with the full length vs truncated IL-3 and GM-CSF proteins. Interestingly, the protein expression or phosphorylation levels were not always decreased by the truncated protein compared to the full length. In some cases, the truncated molecules induced an increase in the protein expression or phosphorylation. These data suggest interesting roles for full length and truncated GM-CSF and IL-3 in both normal and malignant hematopoiesis. Further investigation into the regulation of DPP4, and the roles that full length and truncated factors play during normal and malignant hematopoiesis, are important and will allow for a better understanding of the signficance of DPP4 activity during steady state, stressed, and disease hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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