In Vivo Imaging and Tracking of Defined, Repopulating Human Stem Cell Subsets Using Fluorescence Conjugated Nanoparticles in a Clinically Applicable Ex Vivo Protocol.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3031-3031
Author(s):  
Jesper Bonde ◽  
Dustin J. Maxwell ◽  
David A. Hess ◽  
Ryan Lahey ◽  
Michael H. Creer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
In Vivo Imaging ◽  
Ex Vivo ◽  
Human Cells ◽  
Post Transplantation ◽  
Luminescence Signal ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Novel stem cell-based therapies require new imaging techniques to enable the visualization and tracking of transplanted cells in vivo for evaluation of homing and engraftment parameters. Here we present in vitro and in vivo data on nanoparticle labeling of umbilical cord blood (UCB) CD34+ and lineage depleted HSC subsets, in addition to labeling of CD34+ human bone marrow, G-CSF and AMD-3100 M-PBSC. For these studies, we used a 24 hr. clinically applicable ex vivo labeling protocol including protamine complexed ferrumoxide nanoparticles conjugated to Alexa 647 dye or Alexa 750 (FE-PRO[647] or Fe-Pro[750]). Cell cultivation was carried out using serum free X-Vivo 15 defined medium with 10 ng/ml rhTPO, rhSCF, and Flt-3-ligand on retronectin. Transplantation of FACS sorted 97.5% pure FE-PRO [647] labeled human UCB-derived CD34+ cells into NOD/SCID/B2M null mice resulted in mean engraftment levels of 66.7%+/−1.0% CD45+ human cells, after 8 weeks, as compared to 41.8%+/−20.4% in control mice that received non-loaded cells. These data indicate that the FE-PRO [647] did not compromise the engraftment capacity of the human HSC (p>0.05). Moreover, transplantation of labeled human UCB-derived CD34+ cells into NOD/SCID/B2M null mice for in vivo tracking using flow cytometry and magnetic resonance imaging allowed visualization of the FE-PRO[647] labeled CD34+ cells in the spleen and marrow of the recipients, up to three weeks post transplantation. In spleens, human CD34+ FE-PRO [647]+ levels decreased from 20.6.0+/−13.4% (N=5) one week post transplantation to undetectable levels after three weeks (N=7). The total human CD45+ engraftment as evaluated in total murine marrow was 18.7+/−11.3% (N=7) after three weeks. All animals in the cohort were positive for CD34+ FE-PRO[647]+ engrafted human cells (0.8+/− 0.2 %, N=7). In vivo imaging of animals transplanted with 2–5 x 105 human CD34+ cells (16.8% Fe-Pro[750]+ labeled) was performed using the Kodak 4000 MM multimodal imaging unit in which the luminescence signal arising from the nano-labeled human cells can be precisely localized by overlaying the images with x-ray pictures of the animals. Surprisingly, asymmetric engraftment was repeatedly observed between right leg tibia-femur and left-leg tibia-femur in a cohort of 8 NOD/SCID mice at various timepoints over a total of 20 days after intravenous transplantation. Human engraftment was subsequently confirmed and correlated to the luminescence signal by flow cytometry of the bones and spleens of the imaged animals, at the same timepoints. These data demonstrate that nano-particles can be used to label repopulating human HSC for subsequent in vivo tracking, without toxicity to the engrafting cells. This technique offers new methods to dynamically image the homing and engraftment of purified human hematopoietic stem cells over the initial three weeks post-transplantation, in live animals.

Comparison of Human Stem Cell Homing after Intravenous or Intra-Femoral Transplantation Using Multimodal In Vivo Imaging of Repopulating Human CD34+ Cells Labeled with Far-Red Fluorescent-Conjugated Nanoparticles.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2196-2196
Author(s):  
Jesper Bonde ◽  
David A. Hess ◽  
Dustin J. Maxwell ◽  
Ryan Lahey ◽  
Michael H. Creer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
In Vivo Imaging ◽  
Nano Particles ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cell Homing ◽  
Stem Cell Homing ◽  
Cd34 Cells

Abstract The use of novel nano-sized iron particles and magnetic imaging techniques are ideal for studies of homing and trafficking after labeling and transplantation of long-term repopulating, pluripotent human hematopoietic stem cells (HSC). Whereas the use of luciferase as a reporter for in vivo imaging requires transfection or viral transduction of the target cells to generate a measurable signal, we present an in vivo imaging system based upon the measurement of deep tissue penetrating, near far-red Alexa 750 nm organic dye conjugated to nano-sized ferum oxide particles (FE [750]), transiently introduced into highly purified human hematopoietic stem/progenitor subsets through complexing to the cationic agent protamine sulphate (Pro). Previous results from our group demonstrate that we can track the FE-Pro [750] labeled cells for a minimum of 30 days post transplantation using flow cytometry, before the signal diminishes due to cell division. We used a Kodak 4000MM multimodal imaging unit, which allows a precise anatomical localization of the signal measured through overlaying of the high resolution luminescent profile with x-ray images. NOD/SCID Beta2M null mice were transplanted using intravenous (IV) or intra femural (IF) injection with 1 x 105 or 2 x 105 human cord blood CD34+ cells labeled with the FE-Pro[750] nano particles. The animals were imaged directly after the injections to confirm successful transplantation, and then were subsequently imaged over a period of 8 days (cohort 1), 20 days (cohort 2) or 30 days (cohort 3). At the end point of each time period, animals were sacrificed and flow cytometry was performed to assess and confirm the location of the human engraftment in right and left leg bones as well as in spleens. Our imaging data shows that the human stem cells transplanted IF reside in the injection site for up to 10 days post transplantation, before the dilution of the signal becomes evident, with migration to the spleen at that time point indicating active engraftment, but without noticeable spreading of labeled cells to the non-injected leg. IV injected animals showed an initial strong repopulation of the spleens, with subsequent however asymmetric homing to the femur-tibiae of the legs over 8 days post transplantation, indicating a delayed homing as compared to the more direct IF delivery of the transplantation dose. Flow cytometry results confirmed the asymmetric homing to the femur-tibia bones of IV transplanted animals with one mouse in particular showing a 0.6% CD45+/Fe-Pro[750]low engraftment in the left femur-tibia whereas the right femur-tibia showed a stronger 1.3% CD45+/Fe-Pro[750]low engraftment at day 8. In conclusion, we present a novel system for imaging of human hematopoietic stem cell homing and engraftment post transplantation using dye conjugated nano-particles. This system allow an unprecedented capacity to observe and assess the in vivo dynamics of the engraftment process with high resolution, following intravenous or intrafemoral injection of different purified human stem cell populations.

scholarly journals Functional Studies of Ex Vivo Expanded Hematopoietic Stem Cells in Mice and Monkeys

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1899-1899
Author(s):  
Yu Zhang ◽  
Bin Shen ◽  
Meng Qin ◽  
Zhihua Ren ◽  
Xinxin Ding ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Autologous Transplantation ◽  
Hematopoietic Stem ◽  
Cynomolgus Monkeys ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Bone Morrow

Abstract Hematopoietic stem cell (HSC) transplantation has been widely applied for the treatment of malignant blood diseases. However, obtaining sufficient HLA-matched stem/progenitors for cell transplantation is an obstacle for clinical applications. We reported here that an optimal cytokine cocktail in a modified IMDM basal medium was developed that contained stem cell factor, Flt-3 ligand, thrombopoietin, interleukin 3, G-CSF and GM-CSF. Up to 7.3 folds of expanded CD34+ cells with 66.3% CD34+ of whole cells were obtained after 4 days' culture from human umbilical cord blood. Colony-forming unit (CFU) assays showed that expanded CD34+ cells retained the same renewal ability as the pre-expanded counterparts. To test the repopulating ability of the expanded CD34+ in vivo, sixteen NOD/SCID mice were divided to four groups and injected with saline (group 1), 0.4 million pre-expanded CD34+ cells (group 2), 0.4 million 4-day expanded CD34+ cells (group 3), and 2.9 million expanded CD34+ cells (group 4), respectively. Multi-lineage differentiations in the peripheral blood were assessed by flow cytometry with antibodies against a panel of human cell surface markers. In week 3, human CD34+ cells were decreased below 1% in groups 2 and 3, and 1.717%±0.65% in group 4. Whereas, human CD45+ was increased up to 3.831%±1.54%, 3.108%±1.18% and 10.408%±3.27% for groups 2, 3 and 4, respectively. The other human CD41+, CD71+ and CD15+ were also increased in groups 2-4. No expression of any human cell lineage markers was detected in group 1, indicating that expanded human CD34+ cells possessed the repopulating viability of HSCs in vivo. Furthermore, in week 12, the human CD34+ cells were re-isolated from the bone morrow of the mice (one mouse from each group). The isolated human CD34+ cells were again transfused into new NOD/SCID mice for the secondary transplantation. In week 6, human CD45+, CD15+ and CD19+ were observed from the bone morrow cells of sacrificed mice. On the other hand, human CD45+, CD15+ and CD19+ were also detectable in bone morrow cells for all remaining mice in week 24, suggesting that the expanded CD34+ cells could be successfully engrafted into mice in a long term. In addition, the cytokine cocktail was further evaluated for its safety and efficacy in primates. The CD34+ cells were isolated from the peripheral blood of cynomolgus monkeys and expanded for about 8 folds were obtained on day 9. Harvested CD34+ cells were transducted with the gene of green fluorescent protein (GFP). These cynomolgus monkeys (n=11) were administered with cyclophosphamide via intravenous injection at a dose of 50 mg/kg/day for two days. The myelo-suppressed monkeys were randomly divided into three groups as follows: a control group treated with saline (n=3), a group with autologous CD34- cells (n=3), and a group treated with GFP-labeled, expanded autologous CD34+ cells (n=5), respectively. After autologous transplantation, routine blood tests and flow cytometry analysis were performed to determine the proportion of GFP+ cells in the peripheral blood. The flow cytometry analysis revealed that the white blood cells (WBC), neutrophil (NEU) and platelets (PLT) in peripheral blood of cynomolgus monkeys were completely recovered to the normal levels on days 12, 11 and 10 post autologous transplantation of expended CD34+ cells, respectively. For the control groups, WBC, NEU and PLT returned to the normal on days 22, 22 and 12 for the saline treatment and on days 20, 20 and 12 for the CD34- group, respectively. Similarly, the lymphocytes of cynomolgus monkeys were recovered completely on day 20 post autologous CD34+ cell transplantation compared with the saline control (day 25) and the CD34- group (day 22). On day 30 after the autologous transplantation, the GFP+ ratio in CD45+ populations was around 2% in the peripheral blood. GFP+ cells (ranging from 1.8% to 4.1%) were also detected in bone morrow of cynomolgus monkeys. All primates transplanted with the expanded autologous CD34+ cells have survived for 18 months without any noticeable abnormalities. In conclusion, our results indicate that expanded CD34+ cells can be safely and efficiently used for repopulating stem cell compartment in mice and primates, underscoring the potential applications in the clinic. Furthermore, the results from successful autologous transplantation of cynomolgus CD34+ cells strongly suggest a possible application for personalized treatment of blood diseases. Disclosures Qin: Biopharmagen. corp: Employment. Ren:Biopharmagen corp: Employment.

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.

Soluble Interleukin-6 (IL-6) Receptor With IL-6 Stimulates Megakaryopoiesis From Human CD34+ Cells Through Glycoprotein (gp)130 Signaling

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
Xingwei Sui ◽  
Kohichiro Tsuji ◽  
Yasuhiro Ebihara ◽  
Ryuhei Tanaka ◽  
Kenji Muraoka ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Ex Vivo ◽  
Serum Free ◽  
Human Cd34 ◽  
Receptor Component ◽  
Cd34 Cells ◽  
Interleukin 6 Receptor ◽  
Clonal Cultures

Abstract We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34+ cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34+ cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34+ cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34+ cells were subfractionated into two populations of IL-6R–negative (CD34+ IL-6R−) and IL-6R–positive (CD34+ IL-6R+) cells by fluorescence-activated cell sorting. The CD34+IL-6R− cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34+ IL-6R+cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

Multicolor Flow Cytometry Allows Quantitative Analysis of Signal Transduction in Murine and Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5393-5393
Author(s):  
Tamara Riedt ◽  
Claudia Lengerke ◽  
Lothar Kanz ◽  
Viktor Janzen
Keyword(s):  
Signal Transduction ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Intracellular Signaling ◽  
Hematopoietic Stem ◽  
Specific Antibodies ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract The regulation of cell cycle activity, differentiation and self-renewal of stem cells are dependent on accurate processing of intrinsic and extrinsic signals. Traditionally, signaling pathway activation has been detected by immunobloting using phospho-specific antibodies. However, detection of signal transduction in rare cells within heterogeneous populations, such as hematopoietic stem and progenitor cells (HSC) has been difficult to achieve. In a recently reported approach to visualize signaling in selected single c-Kit+ Sca-1+ Lin− (KSL) bone marrow cells, cells were sorted onto glas slides by flow cytometry and signaling was detected by confocal fluorescence microscopy, a very time consuming method that thus restricts the number of cells that can be analysed simultaneously. Moreover it permits only qualitative, but not quantitative signaling evaluation (Yamazaki et al., EMBO J. 2006). Here, we report a new protocol allowing quantitative measurement of signaling activity in large numbers of defined murine and human hematopoietic cells. The cells are stained with established surface markers and then phospho-specific antibodies are used to detect the levels of active intracellular signaling molecules. Signals are quantified by flow cytometry fluorescence measurement. Importantly, the protocol developed in our laboratory enables preservation of surface marker staining identifying the cells of interest inspite the fixation and permeabilization procedures necessary for intracellular signaling detection. This applies also for antigens previously reported to be particularly vulnerable to standard fixation and permeabilization approaches (e.g. the murine stem cell markers c-Kit and Sca1). Thus, our protocol provides an easy and reliable method for quantifying the activation degree of several intracellular signaling pathways on single cell level in defined hematopoietic (stem) cells within the heterogeous bone marrow (BM) compartment. Using cytokines known to exert a biological effect on HSCs, we have examined the susceptibility of KSL murine BM cells and human BM CD34+ cells to cytokine-induced signaling. We have performed extensive dosage titration and time course analysis for multiple cytokines (SCF, TPO, Flt-3, IL-3, IL-6, Ang-1, SDF-1α, TGF-β, and BMP-4) and signaling pathways (ERK, Akt, p38MAPK, Jak-Stat, TGF-β/BMP-Smad) in murine KSL BM cells. The activation intensity and the duration of signal activity as measured by the expression of corresponding phosphorylated proteins were cytokine specific. The obtained results can be used as a platform to explore signaling alterations in distinct compartments of the hematopoietic system, and may provide mechanistical insights for observed bone marrow defects (e.g impaired ERK signaling pathway has been detected as a possible cause of hematopoietic defects in Caspase-3 mutant murine HSCs, Janzen et al, Cell Stem Cell 2008). Furthermore, we could show that the technique is also applicable to human BM cells and that the human hematopoietic stem cell marker CD34 is also preserved by our fixation and permeabilization protocol. Preliminary results suggest that cytokines induce similar signaling activation in human CD34+BM cells collected from healthy donors. As observed in mouse KSL BM cells, stimulation of human CD34+cells with human stem cell factor (hSCF) induced activation of the ERK but not the Akt pathway. Ongoing experiments analyse the stimulatory effects of other cytokines such as thrombopoietin (TPO) and fms-related tyrosine kinase 3 (Flt-3) and their corresponding pathways. Moreover, comparative studies are underway analyzing cross-reactivity between mouse and human cytokines, aiming to provide insights into cytokine-induced biases in commonly used xenotransplantation models.

Comparative Blood Group Profiling of Human Erythroid Cells (EBs) Generated from Adult Blood (AB), Cord Blood (CB), Human Embryonic Stem Cells (hESC) and Induced Pluripotent Stem Cells (iPS)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1027-1027 ◽  
Author(s):  
Barbara Ghinassi ◽  
Maria Themeli ◽  
Kai-Hsin Chang ◽  
Gregory Halverson ◽  
Ghazala Hashmi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Blood Group ◽  
Ex Vivo ◽  
Antigen Expression ◽  
Band 3 ◽  
Blood Group Antigens ◽  
Dna Genotyping

Abstract Abstract 1027 Red blood cells (RBC) survive shear forces in the microvasculature because trans-membrane complexes embedded in the lipid bilayer attach their membrane to the cytoskeleton assuring its flexibility. The expression of clinically relevant red blood cell antigens present on these complexes is determined by genetic polymorphisms and their developmental regulation. Therefore, flow cytometry studies of blood group antigens may provide insights both on potential immunogenicity and on membrane structure of ex-vivo generated EBs. Blood group antigen profiles of EBs expanded ex vivo from one AB (three experiments), three CB, the H1 hESC line and one iPS line derived from mononuclear cells from a healthy donor were compared by flow cytometry using commercially available antibodies recognizing antigens present on proteins in the 4.1R [Duffy (Fya and Fy3), Kell (Kell prot, K/k, Kpa/Kpb, Jsb) and glycophorin C (GPC, Ge2)] and ankyrin R [glycophorin A (GPA, CD235a, M and EnaFS) RhAG and band 3 (Wrb)] complexes and on other important membrane proteins [glycophorin B (GPB, s and U), urea transporter (Kidd, Jk3), the complement receptor (CD35) and inhibitors of complement-mediated lysis (CD55 and CD59)]. Controls included DNA genotyping (CB, AB and H1-hESC) (HEA-Bead Chip, Immunocor, Norcross, GA) and immunophenotyping of blood red cells from the same AB and CB. Antigen expression similar to that observed on in vivo generated RBC was considered normal. EBs were generated from AB and CB at day 10 in HEMAser cultures whereas EBs from hESC and iPS were derived using previously optimized protocols. The maturation state was determined by morphological analyses and CD36/CD235a profiles. Irrespective of the stem cell source, the immunophenotype of ex-vivo expanded EBs was consistent with that predicted by genotyping. However, source specific differences in the magnitude of antigen expression and in the changes with maturation were observed (see Figure). Immature EBs from AB expressed normal levels of the antigens present on both the 4.1R (Duffy, Kell, GPC) and ankyrin R (GPA, M/N, EnaFS, RhAG and band 3) complexes. With maturation, expression of 4.1R-associated antigens remained normal while that of ankyrin R associated antigens varied (M decreased and RhAG increased). EBs from CB expressed normal levels of antigens present on the ankyrin R complex and of some of those present on the 4.1R complex (Duffy, Kell protein and GPA). However, expression of epitopes on Kell protein varied with some antigens expressed at normal levels (k and Jsb) and others (Kpa/Kpb) at levels 2x greater than normal. With maturation, CB-derived EBs maintained normal levels of ankyrin R associated antigens while those associated with complex 4.1R became barely detectable. EB from hESC expressed unbalanced levels of proteins associated with both ankyrin R (2x levels of GPA and barely detectable levels of RhAG) and 4.1R [3x levels of Duffy and 2x levels of Jsb (Kell) with normal levels of k and Kpb (Kell) antigens] complexes. The variegation in expression of different epitopes on the same protein observed with CB- and hESC-derived EBs likely reflect altered structural conformation of the complexes rather than differences in protein concentration on the membrane. EBs from iPS, as those from AB, expressed normal levels of antigens present on Ankyrin R and 4.1R complexes which increased with maturation. Irrespective of stem cell sources, EBs expressed normal levels of GPB and Kidd. EBs from AB expressed normal levels of the complement regulatory proteins tested which in the case of CD59 CD59 decreased with maturation. EBs from CB expressed normal levels of CD35 and CD59 but 2x levels of CD55 with expression of CD35 and CD55 decreasing with maturation. EBs from iPS expressed 2x levels of CD35 and CD55 and expression of these antigens was not affected by maturation. The observation that blood group antigenic profiles of ex-vivo generated EBs are consistent with those predicted by DNA-genotyping suggests that these cells are unlikely to be immunogenic for known epitopes. However, the antigen profiles of ankyrin R and 4.1R complexes were normal only for AB and iPS-derived EBs raising the possibility that antigenic deviations seen in EBs derived from CB and hESC may have immunologic or functional consequences in vivo. Disclosures: No relevant conflicts of interest to declare.

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.

UM171 Is a Novel and Potent Agonist Of Human Hematopoietic Stem Cell Renewal

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 798-798 ◽  
Author(s):  
Iman Fares ◽  
Jalila Chagraoui ◽  
Yves Gareau ◽  
Stephane Gingras ◽  
Ruel Rjean ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Stem Cell Population ◽  
Fed Batch ◽  
Self Renewal ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
The Impact

Abstract Various combinations of soluble factors have been reported to promote the ex vivo proliferation of human HSCs but few of them are capable of promoting self-renewal. Characterization of StemRegenin (SR1), an aryl hydrocarbon receptor (AhR) antagonist that promotes expansion of CD34+ cells ex vivo, provided a proof of principle that low molecular weight (LMW) compounds have the ability to modulate cellular pathways possibly leading to HSC expansion. In an attempt to identify novel putative agonists of HSC self-renewal, we tested a library comprising > 5,000 LMW molecules in a phenotypical screen based on in vitro expansion of CD34+CD45RA- cells. Eight hits were identified, the majority of which were AhR antagonists. Validation studies identified one hit synthesized in our institute which potently expanded human CD34+CD45RA- cord blood cells ex vivo. Importantly this molecule does not antagonize AhR. Structure activity relationship (SAR) generated a compound called UM171 which is 14-25 folds more potent than the starting molecule with an EC50 of 8-15 nM on CD34+ human cord blood (CB). Using the optimal dose of this analog, CD34+, CD34+CD45RA-, and CD34+CD38-CD90+CD45RA-CD49f+ were expanded 115±15, 62±3 and 121±6 fold respectively over the input values after 12 day culture in fed-batch system. The expansion of these phenotypic HSC populations was enhanced, in an additive manner by including SR1. Washout of UM171 led to a rapid loss of the CD34+CD45RA- population suggesting that its effect is reversible. UM171 is not a mitogen and cannot compensate for the absence of cytokines added to the media (Flt3L, SCF, and TPO). Our cell division studies revealed no effect of the compound on division rate compared to control indicating that UM171 cultured cells retain the CD34+CD45RA- phenotype. Apoptosis and cell phonotype analysis showed significantly less cell differentiation and cell death for UM171 cultured cells compared to controls or SR1 supplemented cultures suggesting that the compound acts by anti-differentiation/apoptosis. Similar to SR1, UM171 treated cells showed a 75±20 fold net increase in the numbers of multilineage colony forming cell (CFU-GEMM) and 150±20 fold when both compounds were present in the 12 day culture.To further understand the impact of UM171 on more primitive cell compartments, we transplanted 100, 500, 1000 and 10,000 uncultured CB CD34+ cells and their progeny after a 12-day culture (with or without UM171) in NSG mice and verified the human hematopoietic reconstitution in their bone marrow 20 weeks later. Mice transplanted with UM171 expanded cells showed a much higher level of human CD45 engraftment compared to uncultured or control expanded cells. The NSG Repopulating Cells (NRC) showed a net expansion ex vivo only in the presence of UM171 with values ranging from 3.1 to 5.5 fold increase over input value in 6 independent experiments. Interestingly, the co-treatment with SR1 did not significantly enhance the impact of UM171 on NRC expansion. In these fed-batch conditions SR1 treatment maintained the NRC population at input values. Importantly, we observed a linear relationship between the number of UM171-treated cells transplanted in vivo and level of human CD45 engraftment. Moreover, at limit dilution, these cells were capable of human myeloid and lymphoid differentiation in NSG BM environment suggesting that UM171 expanded cells remain multipotent. Together, our studies have identified a novel compound which truly expands human progenitors while amplifying the more primitive stem cell population. UM171 has successfully passed toxicity studies and is in the final phase of GMP production for a phase I study which will be launched shortly. In addition, preliminary evidence suggests that UM171 strikingly enhances gene transfer efficiency to primitive human CD34 CB cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Functional Heterogeneity of Human CD34+ Cells Isolated in Subcompartments of the G0 /G1 Phase of the Cell Cycle

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4384-4393 ◽  
Author(s):  
André Gothot ◽  
Robert Pyatt ◽  
Jon McMahel ◽  
Susan Rice ◽  
Edward F. Srour
Keyword(s):  
Cell Cycle ◽  
Ex Vivo ◽  
G1 Phase ◽  
Dna And Rna ◽  
Human Cd34 ◽  
Rna Content ◽  
Cd34 Cells ◽  
Ex Vivo Culture

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34+ cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34+ cells isolated in G0 (G0CD34+ cells) than in those residing in G1 (G1CD34+ cells). However, as MPB CD34+ cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34+ cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34+ cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34+ cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34+ cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2dim population whereby LTHC-IC frequency was higher for CD34+ cells reselected in G0 after in vitro division than for CD34+ cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2bright CD34+ cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.

scholarly journals Development of in Vivo Models of Paediatric Burkitt Lymphoma Allowing Therapeutic Target Analysis and Drug Testing

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5501-5501
Author(s):  
Natalie Bell ◽  
Helen J Blair ◽  
Josef Vormoor ◽  
Chris Bacon ◽  
Vikki Rand ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Cell Lines ◽  
In Vivo Imaging ◽  
Target Selection ◽  
Post Transplantation ◽  
Bioluminescent Signal ◽  
Splenic Enlargement ◽  
Renal Enlargement

Abstract Childhood sporadic Burkitt lymphoma (BL) is a highly aggressive mature B-cell lymphoma. Patients require intensive chemotherapy which results in prolonged periods of hospitalisation for distressing and dangerous side effects. Additionally those with refractory disease or those that relapse have an extremely poor prognosis. As a result, there is an urgent need to develop new targeted treatments that have undergone rigorous pre-clinical evaluation. The aim of this project is to develop improved models for target selection and pre-clinical validation to reduce the failure of translation of novel agents. Two models are being developed. Firstly, xenotransplantation of BL cell lines is being used to optimise transplantation protocols and to provide an orthotopic assay for in vivo functional genomic (RNAi) screening. This is currently being used in genome-wide target identification screens. Secondly, cryopreservation of viable BL blasts is allowing development of a patient-derived xenograft model, suitable for validation of candidate targets by both RNAi and pharmacological inhibition and for testing novel therapeutic agents. The sporadic BL cell lines CA46 and RAMOS have been transplanted into NOD/LtSz-scid IL-2Rg-/- (NSG) and RAG2-/-gc-/- (RAG2) mice at reducing cell doses (105, 104 and 103 cells/mouse). This approach will identify the optimal conditions for orthotopic engraftment. Prior to transplantation, cells were transduced with the lentiviral vector pSLIEW expressing green fluorescent protein (analysis and cell sorting) and firefly luciferase (in vivo bioluminescent imaging). Engraftment was then monitored in real-time using the Caliper in vivo imaging system (IVIS). CA46 and RAMOS cell lines have been transduced with SLIEW, sorted to high purity (>90%) and engrafted in both NGS and RAG2 mice. IVIS imaging of mice transplanted with CA46 cells showed that rate of engraftment was relative to cell dose. All six mice showed a bioluminescent signal over the femurs by 3 weeks post transplantation, indicative of bone marrow (BM) engraftment. Analysis of BM by flow cytometry confirmed engraftment in the two mice analysed, as determined by GFP positivity and presence of the human B-cell markers CD10, CD19 and CD20. Renal enlargement was observed in all mice analysed, with dense infiltration by mature lymphoid blasts evident microscopically. Immunohistochemistry confirmed infiltration with human CD20 positive cells. Splenic enlargement was observed in five out of six mice and engraftment was again confirmed histologically. Onset of disease was rapid. Mice were killed when they showed signs of ill health (see table). A similar pattern of engraftment was observed in NSG and RAG2 mice transplanted with RAMOS cells, although signal intensity was not relative to transplanted cell dose. Bioluminescent signal over the femur was detected in four out of six mice by 3 weeks post transplantation. NSG and RAG2 mice receiving 103 cells, with no IVIS signal, were negative for BM engraftment by flow cytometry. Renal enlargement was not observed, however focal renal lesions were evident macroscopically in three out of six mice. These lesions were composed of human CD20 positive cells. Splenic enlargement was not observed, although a degree of splenic engraftment was identified histologically. Onset of disease was also rapid. Following the initial cell line model development, we are now able to transplant the first patient-derived BL sample. If successful, primograft material will be labelled with luciferase (pSLIEW) and re-transplanted, allowing in vivo imaging. When combined with in vivo RNAi screening, this advanced pre-clinical model for target selection and validation may help reduce target drop-out during early phase clinical trials. This is a critical step, not only to avoid exposing children to ineffectual and potentially toxic drugs, but also to avoid delay in the development of new effective therapies. Abstract 5501. TableCA46DoseSpleenKidneyBMSurvival (days)RAMOSDoseSpleenKidneyBMSurvival (days) RAG2105+++++na29 RAG2105+++++31104+++++++38104-/+-/++29103+++na38103--/+-39 NSG105++na23 NSG105-+++37104++++++31104-/+-/+na30103+++++na38103-++-39 CD20 positivity for spleen and kidney; none (-), < 10% (-/+), 10 - 50% (+), > 50% (++), 100% (+++). For BM engraftment (CD10, CD19 and CD20 positive); no engraftment (-), engrafted (+), not assessed (na). Disclosures No relevant conflicts of interest to declare.

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