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.

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.

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.

A Structure-Function Study of the Activity of Stem Cell Factor in Stimulating the Expansion of Cord Blood CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3501-3501
Author(s):  
Bin Shen ◽  
Wenhong Jiang ◽  
Jie Fan ◽  
Wei Dai ◽  
Xinxin Ding ◽  
...  
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibody ◽  
Stem Cell ◽  
Stem Cell Factor ◽  
Culture Medium ◽  
Cell Number ◽  
Full Length ◽  
Cell Culture Medium ◽  
Cd34 Cells

Abstract Stem cell factor is one of the most important growth factors for human hematopoietic stem cells (HSC). Recombinant human stem cell factor (rhSCF) can stimulate HSC expansion and regeneration in vitro, when it is used in combination with other cytokines like Flt-3L and TPO. However, the specific structural region(s) of the rhSCF protein that are critical for its function in HSC expansion are still unknown. Few studies have addressed this problem, to date. We have recently reported the production of a novel monoclonal antibody (named 23C8) against rhSCF, and the demonstration that 23C8 could inhibit the ability of rhSCF to enhance HSC expansion. Here, we report the identification of a short polypeptide from rhSCF that contains the epitope for binding to 23C8, and, like the full-length rhSCF, is able to stimulate the expansion of umbilical cord blood (UCB)-derived CD34+ cells. Twelve short polypeptides were designed and synthesized, which cover the full length of rhSCF, with 3-5 amino acids overlaps. 23C8 was collected from hybridoma cell culture medium and further purified using protein G affinity chromatography. ELISA was used to identify the polypeptide(s) that positively react with 23C8 among all the synthesized polypeptides. In addition, the effects of the synthetic polypeptides on human HSC expansion capacity were evaluated by supplementing the cell culture medium with 100 ng/ml of a given polypeptide. Total cell number and CD34+ cell number of each group were monitored on day 6. Our novel anti-SCF monoclonal antibody (23C8) partially blocked SCF’s function in human UCB CD34+ cell expansion. Of all the polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 40 to 57, was recognized by 23C8 during ELISA. P0, like the full-length rhSCF, enhanced expansion of CD34+ cells derived from human UCB. P0 addition increased the numbers of total nucleated cells and CD34+ cells by 10.58±0.86 and 4.63±0.43 folds, respectively. For comparison, the extents of increases in cell numbers in the vehicle control group was 3.15±0.99 fold (total nucleated cells) and 1.07±0.11 fold (CD34+ cells), respectively. Residues 40-57 of hrSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells in vitro. The short P0 peptide is a potential candidate for development as a synthetic substitute for rhSCF in clinic applications. Disclosures Jiang: Biopharmagen.corp: Employment. Jiang:Biopharmagen.corp: Employment.

scholarly journals Granulomonocyte-associated lysosomal protein expression during in vitro expansion and differentiation of CD34+ hematopoietic progenitor cells

Blood ◽  
1995 ◽  
Vol 86 (11) ◽  
pp. 4115-4123 ◽  
Author(s):  
C Scheinecker ◽  
H Strobl ◽  
G Fritsch ◽  
B Csmarits ◽  
O Krieger ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Number ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
In Vitro Expansion ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Kinetics Of

Using an in vitro expansion and differentiation system for human CD34+ cord blood (CB) progenitor cells, we analyzed the induction and expression kinetics of the granulomonocyte associated lysosomal proteins myeloperoxidase (MPO), lysozyme (LZ), lactoferrin (LF), and macrosialin (CD68). Freshly isolated CD34+ CB cells were negative for LZ and LF, and only small proportions expressed MPO (4% +/- 2%) or CD68 (3% +/- 1%). Culturing of CD34+ cells for 14 days with interleukin (IL)- 1, IL-3, IL-6, stem cell factor, granulocyte-macrophage colony- stimulating factor (GM-CSF), and G-CSF resulted in on average a 1,750- fold amplification of cell number, of which 83% +/- 7% were MPO+. Without addition of GM-CSF and G-CSF, lower increases in total cell numbers (mean, 211-fold) and lower proportions of MPO+ cells (54% +/- 11%) were observed. The proportion of MPO+ cells slightly exceeded but clearly correlated with the proportion of cells positive for the granulomonocyte associated surface molecules CD11b (Mac-1), CD15 (LeX), CD64 (Fc gamma RI) CD66, or CD89 (Fc alpha R). At day 14 MPO+ and LZ+ cells were virtually identical. However, at earlier time points during culture (days 4 and 7), single MPO+ or LZ+ cell populations were also observed, which only later acquired LZ and MPO, respectively. Maturation of cells into the neutrophilic pathway was indicated by the acquisition of MPO, followed by LZ. In contrast, maturation of cells into the monocytic pathway was indicated by the acquisition of LZ followed by MPO and CD14. CD68 was found to be expressed at day 4 by the majority of cells and was not restricted to the granulomonocytic cells, as cells with megakariocytic (CD41+) or erythroid (CD71hi) features were CD68+. LF expression was observed only in GM- plus G-CSF- supplemented cultures, in which only 26% +/- 5% of cells expressed LF by day 14.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

scholarly journals Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.

1996 ◽  
Vol 183 (3) ◽  
pp. 837-845 ◽  
Author(s):  
X Sui ◽  
K Tsuji ◽  
S Tajima ◽  
R Tanaka ◽  
K Muraoka ◽  
...  
Keyword(s):  
Stem Cell Factor ◽  
Current Data ◽  
Erythroid Cells ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Interleukin 6 Receptor ◽  
Human Sera ◽  
Stimulation Of

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1735-1738 ◽  
Author(s):  
Stephanie Bridenbaugh ◽  
Linda Kenins ◽  
Emilie Bouliong-Pillai ◽  
Christian P. Kalberer ◽  
Elena Shklovskaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Cd8 Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

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