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.

Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1068-1068
Author(s):  
Naoko Takebe ◽  
Thomas MacVittie ◽  
Xiangfei Cheng ◽  
Ann M. Farese ◽  
Emily Welty ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2244-2252 ◽  
Author(s):  
Jean-François Arrighi ◽  
Conrad Hauser ◽  
Bernard Chapuis ◽  
Rudolf H. Zubler ◽  
Vincent Kindler
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Cell Number ◽  
Interleukin 4 ◽  
Flt3 Ligand ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

Ex Vivo Large Scale Generation of Human Platelets from Cord Blood CD34+ Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1892-1892
Author(s):  
Takuya Matsunaga ◽  
Ikuta Tanaka ◽  
Masayoshi Kobune ◽  
Yutaka Kawano ◽  
Maki Tanaka ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Large Scale ◽  
Culture Medium ◽  
Ex Vivo ◽  
Aggregation Assay ◽  
Three Phase ◽  
Cd34 Cells ◽  
Phase Culture

Abstract To obtain a large quantity of platelets (PLTs) from cord blood stem cells (CBSC) in vitro, we employed three-phase culture system. We first expanded CBSC on a monolayer of human telomerase catalytic subunit gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL) and thrombopoietin (TPO) for 14 days (1st phase), and then cultured them to differentiate into megakaryocytes for another 14 days with refreshing medium which contain interleukin-11 (IL-11) in addition to original cytokine cocktail (2nd phase). Subsequently, we transferred the cells to a liquid culture medium containing SCF, FL, TPO and IL-11, and cultured them for 5 days (3rd phase) to recover PLTs in the culture medium. The quantity of PLTs recovered from one CB unit (5 x 106 CD34+ cells) was calculated to be 10.5 units (2 x 1011 PLTs). These CB-derived PLTs exhibited quite similar feature as those from peripheral blood in morphology as revealed by electron micrograph and in functions as revealed by aggregation assay and by FACS detecting expression of P-selectin and activated glycoprotein IIb-IIIa antigens upon fibrinogen/ADP stimulation. Thus our three-phase culture system was considered to be useful for large scale generation of PLTs from CB for clinical usage.

Decrease in Apoptosis and Increase in Polyploidization of Megakaryocytes by Stem Cell Factor During Ex Vivo Expansion of Human Cord Blood CD34+Cells Using Thrombopoietin

Stem Cells ◽  
2002 ◽  
Vol 20 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Jeong-Hae Kie ◽  
Woo-Ick Yang ◽  
Mi-Kyung Lee ◽  
Tae-Jung Kwon ◽  
Yoo-Hong Min ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Stem Cell Factor ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2244-2252 ◽  
Author(s):  
Jean-François Arrighi ◽  
Conrad Hauser ◽  
Bernard Chapuis ◽  
Rudolf H. Zubler ◽  
Vincent Kindler
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Cell Number ◽  
Interleukin 4 ◽  
Flt3 Ligand ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

Experimental attempt to produce mRNA transfected dendritic cells derived from enriched CD34+ blood progenitor cells

Open Medicine ◽  
2008 ◽  
Vol 3 (1) ◽  
pp. 21-28
Author(s):  
Paula Lazarova ◽  
Gunnar Kvalheim ◽  
Liana Gercheva ◽  
Krassimir Metodiev
Keyword(s):  
Prostate Cancer ◽  
Dendritic Cells ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
High Dose ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

AbstractIt Peripheral blood progenitor enriched CD34+ cells (PBPC) are rather often used as stem cell background in cancer patients following high dose therapy. Keeping in mind that precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34+ cells might also serve as starting cells for ex-vivo production of DC. The aim of the present study is to develop a clinical grade procedure for ex-vivo production of DC derived from enriched CD34+ cells. Various concentrations of CD34+ cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-a, SCF, Flt-3L and INF-a. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave equal results as serum-containing medium. Following incubation, the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. The results of our study show that frozen enriched CD34+ cells can be an alternative and efficient source for production of DCs for therapeutic purpose.

scholarly journals Pilot-Scale Production of Megakaryocytes/Platelets from Cord Blood CD34+ Cells in a Bottle Turning Device Culture System

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 819-819
Author(s):  
Xin Guan ◽  
Lan Wang ◽  
Yu Zhang ◽  
Bin Shen ◽  
Zhihua Ren ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Pilot Scale ◽  
Cell Number ◽  
Human Platelets ◽  
Scale Production ◽  
Culture Bottle ◽  
Cd34 Cells ◽  
Pilot Scale Production

Abstract Due to a donor-platelet shortage, ex vivo generated megakaryocytes/platelets have emerged as an effective substitute to alleviate thrombocytopenia. We have previously reported a "two-stage culture system" for producing megakaryocytes/platelets from CD34+ cellsand demonstrated their safety and efficacy in myeloablative murine and non-human primate models. Here, we present pilot-scale production of megakaryocytes/platelets in a bottle turning device culture system that significantly increases the yield of functional megakaryocytes/platelets. Enrichedcord blood CD34+ cells were first cultured in modified IMDM basal medium (serum-free) with the addition of stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), and interleukin (IL)-3 in a T-25 flask. After 6 days of expansion, cells were transferred into a 2 L culture bottle with 600 ml medium in a bottle turning device at the density of 1.5×105/mL for additional 7 days. The culture medium was modified IMDM supplemented with SCF, TPO, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). At various time points, cell numbers were counted with a hemocytometer and expression of CD34, CD41a, and CD42b surface markers was monitored by flow cytometry. Resulting megakaryocytes were further confirmed by morphological examination and DNA ploidy analysis. Expression of relevant oncogenes was measured by quantitative real-time polymerase chain reaction (qPCR) for evaluating potential tumorigenicity. Moreover, these megakaryocytes were transplanted into sub-lethally irradiated NOD/SCID mice (1×106 CD41a+/CD42b+ megakaryocytes per mouse) to assess human platelet-releasing potential and function in vivo. The initial cell number was 1×106 with CD34 purity of 93.4% ±2.8%. For 6-day culture, the absolute number of total cells and CD34+ cells reached 9.3×107±1.8×107 and 4.0×107±5.6×106, respectively, which were calculated into a 92.9±18.1 and 42.8±5.2-fold increase, respectively. For additional 7-day culture in the turning bottles, 2.2×1010±3.2×109 cells were obtained with the proportions of CD41a+ and CD42b+ cells at 80.1%±7.4% and 63.6%±5.9%, respectively. Approximately 2.0×104 megakaryocytes/platelets were generated from each input CD34+ cell. These megakaryocytes were morphologically discernible as they were much larger than starting CD34+ cells with apparent lobular nuclei and numerous α-granules. DNA content analysis revealed that about 25.4%±1.3% of megakaryocytes exhibited a polyploidy level of >8N. Moreover, about 10% megakaryocytes have formed pro-platelet fragments and released platelets in cultures. Compared to initial CD34+ cells, generated megakaryocytes showed similar levels of expression of proto-oncogenes including c-Myb, N-Ras, and K-Ras whereas c-Myc, bmi-1, cyclin B, and hTERT expression was decreased, strongly suggesting that ex vivogenerated megakaryocytes are unlikely tumorigenic. Releasing of human platelets by injected megakaryocytes were detected in mouse peripheral blood with a percentage of 0.5%±0.1% as early as 30 min following transplantation, after which the percentage of human platelets increased rapidly with a peak level of 3.2%±0.6% at about 4-6 h post-transplantation. The half-life of released platelets in mice was about 10 h and about 23±6 human platelets were released per megakaryocyte in the transplanted mice. Furthermore, these platelets were activated, expressing CD62P after stimulation by ADP, suggesting that released human platelets in mice are functional. Combined, we have established a bottle turning device culture system for pilot-scale production of megakaryocytes/platelets ex vivo. One unit of cord blood (80 ml) with 2×106 CD34+ cells can generate 4×1010 megakaryocytes/platelets that are sufficient for treating 130 patients (with an average weight of 60 kg and infusion cell number 5×106cells/kg). In conclusion, our new culture system is capable of manufacturing GMP-grade megakaryocytes/platelets sufficient for various clinical applications. Disclosures Ren: Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.

Short Pulse of a Small Peptide Agonist of Stromal Cell-Derived Factor - 1 (SDF-1) Enhances the Engraftment of Expanded Human Cord Blood Hematopoietic Progenitor Cells in NOD/SCID Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 404-404 ◽  
Author(s):  
Karen Kwai Har Li ◽  
Carmen K.Y. Chuen ◽  
Shuk Man Lee ◽  
Donald Wong ◽  
Ahmed Merzouk ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Short Pulse ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Peptide Agonist

Abstract SDF-1 is the ligand to the chemokine receptor CXCR-4. A small synthetic peptide agonist of SDF-1 (CTCE-0214) has been shown to expand human cord blood hematopoietic stem and progenitor cells. In this study, we investigated whether a brief exposure of expanded cord blood hematopoietic cells to CTCE-0214 can improve engraftment of the cells into NOD/SCID mice. Published in vivo studies demonstrated that the administration of CTCE-0214 to transplanted NOD/SCID mice mobilized human colony forming cells (CFC) and enhanced human thrombopoiesis (Exp Hematol 32, 300, 2004). Our earlier study showed that CTCE-0214 added to single factors of thrombopoietin (TPO), stem cell factor (SCF), or Flt-3 ligand (F3L) synergistically increased the survival of enriched cord blood CD34+ cells (Blood 102, 960a, 2003). In this study, we further investigated the effects of CTCE-0214 on the ex vivo expansion of CD34+ cells to multi-lineage progenitors and the homing and engraftment capacity of expanded human progenitor cells after a short in vitro exposure to the peptide prior to infusion into NOD/SCID mice. Enriched CD34+ cells (MACS) derived from cord blood were cultured for 8 days in serum-free medium QBSF-60 containing TPO (50 ng/ml), SCF (50 ng/nl) and F3L (80 ng/ml) (TSF), with or without CTCE-0214 (0.01 ng/ml) (TSF+CTCE-0214) added at day 4. Progenitor cells expanded for 8 days in the absence of CTCE-0214 were pulsed with the peptide (100 ng/ml) for 4 hours (TSFpCTCE-0214). Results are summarized in Table. CTCE-0214 significantly (N=30, p≤0.05, paired t-test) increased the fold expansion of total nucleated cells (TNC), CD34+ cells, CD34+CD38- cells, CFU-GM, CFU-E, and CFU-MK (total CFC). Expanded progenitor cells (with and without CTCE-0214) were then infused into irradiated NOD/SCID mice. After 6 weeks, enhanced engraftments of human CD45+ cells (p≤0.05, N=21) were demonstrated in the bone marrow (BM) of mice that received cells cultured in TSF+CTCE-0214. Interestingly, a short pulse of cells expanded in TSF to CTCE-0214 for 4 hours also significantly increased the NOD/SCID engraftment (N=18), although no major changes to the in vitro read-out parameters were observed. The mechanism could be associated with the increased homing capacity of progenitor cells after pulsing with CTCE-0214. In conclusion, our results showed that CTCE-0214 enhances the proliferation of early progenitor cells in culture and exposure to the peptide can enhance the engraftment potential of expanded cells in NOD/SCID mice. The SDF-1 peptide agonist could be developed for application to hematopoietic stem cell transplantation and ex vivo expansion. NOD/SCID Engraftment of Expanded Cord Blood Stem Cells TSF TSF+CTCE-0214 TSFpCTCE-0214 *Fold expansion (mean±SE); **% human CD45+ cells in BM of mice TNC* 84.6±10.4 123.5±15.3 88.5±11.2 CD34+* 8.5±1.3 14.1±2.1 9.6±1.6 CD34+CD38−* 24.6±4.8 48.7±8.6 27.5±5.3 Total CFC* 46.9±6.5 87.9±10.7 50.6±6.4 NOD/SCID** 2.8±0.9 6.7±2.5 8.3±4.0

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.

Towards the Development of a Closed, Nanofiber-Based Culture System for Clinical Expansion of Cord Blood-Derived CD34+ Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4411-4411
Author(s):  
Stephen E Fischer ◽  
Yiwei Ma ◽  
Caitlin Smith ◽  
Anirudhasingh Sodha ◽  
Yukang Zhao
Keyword(s):  
Bone Marrow ◽  
Tissue Culture ◽  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cell Phenotype ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 4411 Interest in ex vivo hematopoietic stem and progenitor cell (HSPC) expansion has increased in recent years due to the growing importance of these cells in the treatment of a variety of both malignant and non-malignant diseases. Ex vivo expansion of cord blood-derived cells has been particularly investigated because cord is a valuable and readily available source of HSPCs, yet contains limited numbers of cells in each unit. Despite these efforts, most attempts to use expanded cord blood HSPCs in the clinic have been unsuccessful due to the generation of insufficient numbers of cells with the appropriate phenotype and the ability to function in vivo. In many ex vivo culture systems, HSPCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSPCs with a substrate. In the natural bone marrow microenvironment, HSPCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D nanofiber-based cell culture substrate (NANEX™). The functionalized NANEX™ substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSPC proliferation while minimizing differentiation. Here, we present our recent work towards developing a closed, NANEX™-based platform for large-scale clinical expansions of cord blood-derived CD34+ cells. We demonstrate that NANEX™ expands CD34+ cells from cord an average of more than 150-fold in 10 day culture, which is at least 2-fold higher than that obtained in standard tissue culture plates. Additionally, we show an approximately 1.5-fold higher proliferation of colony forming cells and a significantly higher engraftment rate in NSG mice for NANEX™-expanded cells compared to cells cultured in tissue culture plates. Furthermore, we demonstrate that the NANEX™ scaffold maintains its HSPC growth promoting characteristics after processing into a closed culture system and offers significant advantages over other culture platforms typically used for HSPC expansions in the clinic (culture bags and T-flasks). Our data indicates that NANEX™ technology provides a robust ex vivo expansion of cord blood HSPCs and, with further development, offers great potential for clinical applications requiring large numbers of functional cells. Disclosures: No relevant conflicts of interest to declare.

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