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.

Efficient and Durable Gene Marking of Hematopoietic Progenitor Cells in Nonhuman Primates After Nonablative Conditioning

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2271-2286 ◽  
Author(s):  
M. Rosenzweig ◽  
T.J. MacVittie ◽  
D. Harper ◽  
D. Hempel ◽  
R.L. Glickman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Blood Stem Cells ◽  
High Level

Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 μg/kg) and G-CSF (20 μg/kg) for 7 days and autologous CD34+ peripheral blood stem cells harvested by leukapheresis. CD34+ cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% ± 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% ± 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naı̈ve (CD45RA+and CD62L+) CD4+ and CD8+cells were the predominant phenotype of the marked CD3+ T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naı̈ve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.

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.

Functional Evaluations of Ex Vivo Induced Endothelial Progenitors for Autologous Transplantation in Non-Human Primates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4302-4302
Author(s):  
Meng Qin ◽  
Xin Guan ◽  
Yu Zhang ◽  
Qing-yu Zhang ◽  
Wei Dai ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Autologous Transplantation ◽  
Double Staining ◽  
Cynomolgus Monkeys ◽  
Injury Model ◽  
Cd34 Cells

Abstract It is possible to treat ischemia and hemophilia A diseases by producing sufficient functional human endothelial progenitor cells (EPCs)/endothelial cells (ECs) in vitro, for use with cell therapy in the clinic. We have previously reported the ability to produce FVIII-secreting EPCs/ ECs derived from human cord blood CD34+ cells. About 1412±102 fold expansion over initial EPCs was achieved after culturing for 21 days. An acute liver sinusoidal endothelial cells (LSEC) injury model in NOD/SCID mice was also developed to verify the functional migrating ability of the generated EPCs/ ECs in vivo. Here, we further applied this culturing technique to expand and subsequently differentiate CD34+ cells into the EPCs/ ECs derived from mobilized peripheral bloods of both human and cynomolgus monkeys. In brief, the CD34+ cells were isolated from human peripheral bloods or from monkeys (n=10) mobilized with human G-CSF/SCF. In the first 6 days, the isolated CD34+ cells were expanded in modified IMDM medium supplemented with human cytokine combinations of SCF, Flt-3L, TPO, IL-3, GM-CSF, and VEGF. From days 7 to 36, the adhering EPCs/ ECs were subsequently differentiated in EBM-2 basal medium with 20% FBS and endothelial growth factors of VEGF、IGF、EGF、FGF, and fibronectin. The purities and phenotypes of the induced EPCs/ECs were assessed in vitro by antibodies against human CD31, vWF, and FVIII for the human or Dil- acetylated- low density lipoprotein (ac-LDL) and FITC-lectin double staining for the monkey cells.In addition, the safety and efficacy of the induced monkey EPCs/ECs was determined in vivo by autologous transplantation in monkey LSEC injury model, which was induced by a toxic agent, monocrotaline (MCT), to disrupt the sinusoidal endothelial barrier and stimulate the incorporation of transplanted cells into liver parenchyma. In the transplantation group (n=7), each monkey was injected with double labeled autologous EPCs/ECs preparations (2×108 cells/500μl in saline), whereas in the control group (n=3) was injected with the same volume saline via hepatic portal vein injections. The cross-sections (20µm in depth) of fixed hepatic tissues were analyzed for grafting and functional migration of transplanted EPCs/ECs. The transplanted cells were identified by lenti-viral gene expressed with green fluorescent protein (red) or direct observation using anti-monkey IgG -microbead- FITC conjugates (green). For in vitro induced EPCs/ECs derived from human peripheral blood cell, the expansion of 834.58±119.03 fold was achieved from the CD34+/VEGFR2+ EPCs on day 21. Total more than 2x 108 FVIII-producing EPCs / ECs were produced from one collection of human peripheral blood (250 mL). On the other hand, the CD34+/VEGFR2+ EPCs (3.6×104 ±2.1×103) from one collection of monkey peripheral blood (20ml) were expanded up to 1274±166 fold and 7211±372 fold on days 24 and 36, respectively (n=4). The EPCs were reached at a logarithmic growth from days 12 to 45. The induced cells can be frozen and resuscitated during any stage of the culturing process. The formation of EC tubes was observed from day 24. Over 80% of expanded cells were EPC/EC-specific and identified by Dil-ac-LDL and FITC-lectin double staining on day 36. All monkeys recoveredfrom the surgeries of portal vein injection and resumed normal diet and behavior after autologous transplantation with cultured EPCs/ECs. Similarly, the routine blood analysis and liver functional enzymes were at the normal level, and no other apparent side effects were observed. About 3.2±1.4% and 2.1±1.1% of liver cells were observed as Dil-ac-LDL and FITC-lectin double positive in the liver cryosections (25 sections per monkey) on days 7 and 14, respectively, indicating that autologous transplanted EPCs/ECs were capable of repopulating into functional ECs in vivo. Furthermore, the injected EPCs/ECs were scattered in the intercellular spaces of hepatocytes at the hepatic tissues on day 14, suggesting that the transplanted cells could migrate towards injured LSEC sites and reconstitute structurally the sinusoidal endothelial compartment in monkey livers. In summary, the large-scale EPCs/ECs were produced from CD34+ cells of both human and monkey peripheral bloods in vitro. The safety and functions of the EPCs/ECs were confirmed in mice and cynomolgus monkeys, strongly suggesting the potential application of these FVIII-producing EPCs/ECs to future clinical study. Disclosures Qin: Biopharmagen. corp: Employment.

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.

Blood CD34 Levels as a Predictor of Successful Peripheral Blood Stem Cell Mobilization In Consecutive Autologous Stem Cell Apheresis Patients From a Single Transplant Center

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4440-4440
Author(s):  
Sujatha Mogili ◽  
Rui Chen ◽  
Michael Becker ◽  
Faith Young ◽  
Gordon L. Phillips ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Autologous Transplantation ◽  
Operating Characteristics ◽  
Hematopoietic Stem ◽  
Prior Chemotherapy ◽  
Cd34 Cells ◽  
Stem Cell Collection

Abstract Abstract 4440 Background: The optimal strategy to mobilize hematopoietic stem cells into peripheral blood for collection has not been defined, and some patients do not successfully mobilize. Failure to harvest the desired number of CD34+ cells results in emotional disappointment for the patient and ineffective utilization of resources. In this study we analyzed various factors influencing CD34+ yields during stem cell collection in patients being considered for autologous transplantation whose peripheral blood CD34+ analysis triggered stem cell collection (usually >10/μ L), and we determined thresholds for successful collection in a single apheresis session. Method: Retrospective chart review of 244 consecutive patients who underwent stem cell collection at the University of Rochester between 2005 and 2008 was conducted. Cells were collected via Cobe semi-automated protocols. For each of the patients, diagnosis, age, gender, type of infusion, marrow status, number of prior chemotherapy treatments, mobilization regimen, CD34+ count/μ l on first day, CD34+/kg after first apheresis, total time of apheresis and volume processed (liters) were recorded. The various mobilization regimens utilized were 1)GCSF, 2)CHEMO (salvage therapy with DHAP, ESHAP, RICE) with GCSF, 3)cyclophosphamide with GCSF, 4)AMD3100 (Plerixafor) with GCSF, and 5) GCSF with GMCSF. The marrow status was defined as no involvement, mild involvement (10-20%), and moderate involvement (>20%). Another factor considered was the number of prior chemotherapy regimens, and the total number of apheresis cycles required to reach targeted yields was recorded. All statistical analyses were conducted using SAS 9.2. All tests were two-sided with p-values ≤ 0.05 considered significant. Result: Analysis revealed a positive linear relationship between the log of initial CD34+ counts and the log transformed number of CD34+ cells/kg on the first day of apheresis (R2= 0.57, p < 0.001). Multivariate analysis suggested that both CD34+ count per μ L blood prior to first apheresis (p < 0.001) and time on the machine (p = 0.08) were positively associated with CD34+ end yield after adjusting for age and gender. A similar analysis on all the mobilization regimens revealed statistical significance for a higher initial CD34 cell count/μ l predicting a higher apheresis yield (p < 0.001). Also, for each of the major diagnoses (AL, HL, MM. NHL), there was a positive relationship between peripheral CD34+ cells/μ l prior to apheresis and CD34+ cells/kg after first apheresis yield (p < 0.001) Another objective of this study was to see if there is a threshold number of CD34+ cells/μ l blood that would predict for lymphoma patients (combined HL, NHL) that at least 4 × 106 CD34+ cells/kg would be reached on the first day of apheresis, and a similar threshold for the multiple myeloma patients (MM) to reach 6 × 106 CD34+ cells/kg on first day. Receiver operating characteristics (ROC) curves were used to determine an optimal initial CD34+ count cutoff and the odds ratio of achieving such a threshold between the two groups was assessed using logistic regressions. Based on the cutoff of 48.3 cells/μ L, lymphoma patients were classified into two groups depending on their initial CD34+ count greater or less than this cutoff. The odds of reaching at least 4 × 106 CD34+ cells/kg on the first day of apheresis when the initial CD34+ count was greater than 48.3 cells/μ L were 24.4 times than those with the initial CD34+ count less than the cutoff (p<0.001). Similarly, for multiple myeloma patients, to reach a threshold of 6×106 CD34+ cells/kg on the first day of apheresis, a cutoff of 90.5 cells/μ L was established. The odds of reaching at least 6×106 CD34+ cells/kg on the first day of apheresis with initial CD34+ count greater than 90.5 cells/μ L were 19.3 times than those with initial CD34+ count less than the cutoff (p<0.001) Conclusion: This study confirmed that the initial peripheral CD34+ count was a good predictor of the CD34+ cells/kg yield on the first day of apheresis, irrespective of disease type or mobilization regimen utilized. A threshold of 48.3 CD34+ cells/μ L for lymphoma patients and 90.5 CD34+ cells/μ L for myeloma patients was established to reach goals of least 4 × 106 CD34+ cells/kg and 6 × 106 CD34+ cells/kg respectively with a single apheresis. These thresholds or others calculated similarly for other collection goals could potentially aid in coordination of apheresis resource utilization. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lower Hematopoietic Progenitor Cell Counts and Yields at Subsequent Donations Is Influenced By a Shorter Inter-Donation Interval between the First and Subsequent Mobilizations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1962-1962
Author(s):  
Sandhya R. Panch ◽  
Brent R. Logan ◽  
Jennifer A. Sees ◽  
Bipin N. Savani ◽  
Nirali N. Shah ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Time Interval ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Peripheral Blood Cd34 ◽  
Cell Counts ◽  
Cd34 Cells

Introduction: Approximately 7% of unrelated hematopoietic stem cell (HSC) donors are asked to donate a subsequent time to the same or different recipient. In a recent large CIBMTR study of second time donors, Stroncek et al. incidentally found that second peripheral blood stem cell (PBSC) collections had lower total CD34+ cells, CD34+ cells per liter of whole blood processed, and CD34+ cells per kg donor weight. Based on smaller studies, the time between the two independent PBSC donations (inter-donation interval) as well as donor sex, race and baseline lymphocyte counts appear to influence CD34+ cell yields at subsequent donations. Our objective was to retrospectively evaluate factors contributory to CD34+ cell yields at subsequent PBSC donation amongst NMDP donors. Methods. The study population consisted of filgrastim (G-CSF) mobilized PBSC donors through the NMDP/CIBMTR between 2006 and 2017, with a subsequent donation of the same product. evaluated the impact of inter-donation interval, donor demographics (age, BMI, race, sex, G-CSF dose, year of procedure, need for central line) and changes in complete blood counts (CBC), on the CD34+ cell yields/liter (x106/L) of blood processed at second donation and pre-apheresis (Day 5) peripheral blood CD34+ cell counts/liter (x106/L) at second donation. Linear regression was used to model log cell yields as a function of donor and collection related variables, time between donations, and changes in baseline values from first to second donation. Stepwise model building, along with interactions among significant variables were assessed. The Pearson chi-square test or the Kruskal-Wallis test compared discrete variables or continuous variables, respectively. For multivariate analysis, a significance level of 0.01 was used due to the large number of variables considered. Results: Among 513 PBSC donors who subsequently donated a second PBSC product, clinically relevant decreases in values at the second donation were observed in pre-apheresis CD34+ cells (73.9 vs. 68.6; p=0.03), CD34+cells/L blood processed (32.2 vs. 30.1; p=0.06), and total final CD34+ cell count (x106) (608 vs. 556; p=0.02). Median time interval between first and second PBSC donations was 11.7 months (range: 0.3-128.1). Using the median pre-apheresis peripheral blood CD34+ cell counts from donation 1 as the cut-off for high versus low mobilizers, we found that individuals who were likely to be high or low mobilizers at first donation were also likely to be high or low mobilizers at second donation, respectively (Table 1). This was independent of the inter-donation interval. In multivariate analyses, those with an inter-donation interval of >12 months, demonstrated higher CD34+cells/L blood processed compared to donors donating within a year (mean ratio 1.15, p<0.0001). Change in donor BMI was also a predictor for PBSC yields. If donor BMI decreased at second donation, so did the CD34+cells/L blood processed (0.74, p <0.0001). An average G-CSF dose above 960mcg was also associated with an increase in CD34+cells/L blood processed compared to donors who received less than 960mcg (1.04, p=0.005). (Table 2A). Pre-apheresis peripheral blood CD34+ cells on Day 5 of second donation were also affected by the inter-donation interval, with higher cell counts associated with a longer time interval (>12 months) between donations (1.23, p<0.0001). Further, independent of the inter-donation interval, GCSF doses greater than 960mcg per day associated with higher pre-apheresis CD34+ cells at second donation (1.26, p<0.0001); as was a higher baseline WBC count (>6.9) (1.3, p<0.0001) (Table 2B). Conclusions: In this large retrospective study of second time unrelated PBSC donors, a longer inter-donation interval was confirmed to be associated with better PBSC mobilization and collection. Given hematopoietic stem cell cycling times of 9-12 months in humans, where possible, repeat donors may be chosen based on these intervals to optimize PBSC yields. Changes in BMI are also to be considered while recruiting repeat donors. Some of these parameters may be improved marginally by increasing G-CSF dose within permissible limits. In most instances, however, sub-optimal mobilizers at first donation appear to donate suboptimal numbers of HSC at their subsequent donation. Disclosures Pulsipher: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Honoraria. Shaw:Therakos: Other: Speaker Engagement.

The Impact of Different Mobilization Strategies in the Setting of Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3254-3254
Author(s):  
Francesco Mazziotta ◽  
Gabriele Buda ◽  
Nadia Cecconi ◽  
Giulia Cervetti ◽  
Lorenzo Iovino ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Stem Cell ◽  
Partial Response ◽  
Peripheral Blood ◽  
High Dose ◽  
Roc Curve Analysis ◽  
Cd34 Cells ◽  
Significant Difference ◽  
The Impact

INTRODUCTION Multiple myeloma (MM) is considered an incurable disease. Despite the introduction of novel agents allowed deeper response, high-dose chemotherapy and autologous stem cell transplantation (ASCT) remain the standard of care for patients (pts) in good clinical conditions. The most used strategies to mobilize stem cells from bone marrow (BM) into peripheral blood are high-dose cyclophosphamide (HD-CTX) plus G-CSF and G-CSF plus plerixafor (G-CSF+P). The goal of this retrospective study is to investigate whether the two different mobilization strategies have an impact on the clearance of monoclonal PCs in the apheresis products and on pts' outcome. PATIENTS AND METHODS We analyzed 62 pts (median age 61, range 41-75, 37 males and 25 women) diagnosed with MM and treated with ASCT between Mar 2014 and Mar 2018 at our Hematology Division (Pisa, Italy). All pts received induction therapy with at least 4 cycles of bortezomib, thalidomide and dexamethasone (VTD). 9/62 pts obtained a less than partial response (PR) and received lenalidomide-based regimens. After induction, 8 (12,9%) pts achieved complete remission (CR), 26 (41,9%) were in PR, 28 (45,2%) obtained a very good partial response (VGPR). 43/62 fit pts received HD-CTX (2-3 g/sqm) on day 1 followed by G-CSF (30 MU/day) started on day 4 until day 7, increased to 60 MU/day from day 8 until the end of apheresis. In 19/62 pts, after 4 days of G-CSF (60 MU/day) administration and not sufficient mobilization, we added plerixafor (0,24 mg/kgbw) for up to 4 consecutive days. In 43/62 pts we collected apheresis samples (10μl) analyzed through flow citometry to enumerate clonal residual PCs. The panel used to asses clonality included: CD138 Per-Cp, CD38 APC, CD19 PE-Cy7, CD45 APC-Cy7, cytoplasmic immunoglobulin K chain and L chain. RESULTS At the end of the peripheral blood stem cell (PBSC) collection, pts treated with HD-CTX presented a higher CD34+ absolute count (p=0.0489) and achieved the threshold of 5x106 CD34+ cells/kgbw in a significantly (p=0.006) higher percentage. We found a nearly significant (p=0.0517) lower count of CD34+ PBSCs in pts who received lenalidomide-based regimens before the mobilization. Performing flow citometry on apheresis samples, we observed that the number of the harvested clonal PCs showed a significant correlation (p=0.0115) with the occurrence of post-ASCT relapse. ROC curve analysis investigating the predictive effect of the number of pathological PCs on disease relapse showed an area under the curve of 0,6978 (95% CI 0.5392-0.8564; p=0.0267). Neither BM residual PCs detectable on BM biopsies performed before apheresis (r=-0.1323; p=0.609) nor the type of mobilization scheme (p=0.707) had an impact on the proportion of clonal PCs in the graft. Additionally, we did not observe any statistically significant difference in progression free- (PFS) (p=0.8276) and overall survival (OS) (p=0.2475) between the HD-CTX and G-CSF+P groups. DISCUSSION PBSC mobilization has a succession rate > 85%. Despite the use of HD-CTX to increase PBSC yields and decrease tumor burden, there is not clear evidence of a superior mobilization strategy. Additionally, HD-CTX has a not negligible toxicity and approximately 10% of the pts require hospitalization. Conversely, G-CSF+P is a safe and effective approach also in poor mobilizers. In our study, we observed a significative difference in the apheresis yields (p=0.0489) and in the percentage of pts who achieved the threshold of 5x106 CD34+ cells/kgbw (p=0.006) in favor of HD-CTX. Additionally, the detection of harvested residual clonal PCs could be a promising strategy to recognise pts more likely to relapse after ASCT. Nonetheless, we failed to demonstrate a superior effect of HD-CTX in the clearance of harvested clonal PCs, in agreement with the absence of a different pts' outcome amongst the two mobilization strategies. In conclusion, the choice between the two regimens is challenging and requires careful consideration of multiple factors. Overall, young fit pts, especially in the high-risk setting, should be treated with all appropriate modalities including chemiomobilization followed by double-ASCT. Conversely, in pts candidate to a single-ASCT it is reasonable to use G-CSF+P, since HD-CTX does not improve PFS and OS and add toxicity. The absence of an in-vivo purging effect on apheresis products of chemiomobilization further strengthens a chemotherapy-free mobilization. Disclosures Galimberti: Roche: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

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