A Small Animal In Vivo Imaging Model of Expanded vs. Unexpanded UCB Stem Cells Reveals Differential Patterns of Engraftment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1191-1191
Author(s):  
David Steiner ◽  
Simon N. Robinson ◽  
William K. Decker ◽  
Frank C. Marini ◽  
Elizabeth J. Shpall ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vivo Imaging ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Small Animal ◽  
Firefly Luciferase ◽  
Pathologic Evaluation ◽  
Imaging Model ◽  
Cd34 Cells

Abstract INTRODUCTION: Use of HLA-mismatched umbilical cord blood (UCB) allows transplant of patients who lack related HLA identical or matched unrelated donors. A major disadvantage of UCB is the limited number of stem cells available within the graft, a contributor to delayed engraftment, especially in adults. Two strategies have been adopted clinically in an attempt to overcome this barrier. 1. ex vivo expansion of some or all of critical graft subfractions; 2. transplantation of two different UCB grafts. These two strategies are not mutually exclusive and are also sometimes used in combination. Little is known about the basic biology that underlies the differential migration and homing of the graft components. To study the biology of complex UCB transplants, we have developed a small animal in vivo imaging model. METHODS: CD34-selected UCB cells were transduced overnight with a lentiviral construct consisting of GFP or a firefly luciferase/GFP fusion. After 24 hours, SCF, FLT3, G-CSF, and thrombopoietin were added and the cells were cultured for 3 to 8 days. All cultured cells were then injected into non-lethally irradiated, immunocompetent mice (NOD/SCID/IL-2Rγ − /−). Transduced cells were tracked weekly by bioluminescent imaging. RESULTS: On culture day 3, >99% of cells remained CD34+ and no expansion was observed. After expansion in culture between days 3 and 8, 19% +/− 18.5% of transduced cells were GFP+CD45+ (n= 8). Cells cultured for three days (non-expanded) (3-11x105 CD34+ cells/3-8.6x104 GFP+) were able to create a detectable engraftment signal by post-transplant day 8. The engraftment signal was delayed until day 10 when an 8-fold excess of the expanded cultured cells (4.2x106 total consisting of 1.21x106 CD34+ cells/2.3x105 GFP+ and 6.51x105 CD34-GFP+ cells) were injected. Long term engraftment (>30 days) was unaffected by expansion in culture. However, expanded UCB exhibited a differential pattern of homing and engraftment in comparison to unexpanded UCB. Mice receiving unexpanded UCB exhibited a strong engraftment signal from the area of the calvarium in addition to signals from other marrow spaces. The calvarium signal was only weakly and intermittently observed among mice who received the expanded UCB graft. Pathologic evaluation of these areas is in progress. CONCLUSIONS: 1. Expanded cells were capable of supporting engraftment, though at a slower rate than their unexpanded counterparts. This delay might ultimately be addressed through the optimization of the expansion technique. 2. The physiologic significance of the differential engraftment pattern(s) is unclear and will require further study. Figure Figure

scholarly journals In Vivo Imaging of Peripheral Benzodiazepine Receptors in Mouse Lungs: A Biomarker of Inflammation

2005 ◽  
Vol 4 (4) ◽  
pp. 7290.2005.05133 ◽  
Author(s):  
Matthew J. Hardwick ◽  
Ming-Kai Chen ◽  
Kwamena Baidoo ◽  
Martin G. Pomper ◽  
Tomás R. Guilarte
Keyword(s):  
In Vivo Imaging ◽  
Ex Vivo ◽  
Inflammatory Diseases ◽  
Imaging Techniques ◽  
Benzodiazepine Receptors ◽  
Small Animal ◽  
Small Animal Pet ◽  
Planar Imaging ◽  
Peripheral Benzodiazepine Receptors

The ability to visualize the immune response with radioligands targeted to immune cells will enhance our understanding of cellular responses in inflammatory diseases. Peripheral benzodiazepine receptors (PBR) are present in monocytes and neutrophils as well as in lung tissue. We used lipopolysaccharide (LPS) as a model of inflammation to assess whether the PBR could be used as a noninvasive marker of inflammation in the lungs. Planar imaging of mice administrated 10 or 30 mg/kg LPS showed increased [123I]-( R)-PK11195 radioactivity in the thorax 2 days after LPS treatment relative to control. Following imaging, lungs from control and LPS-treated mice were harvested for ex vivo gamma counting and showed significantly increased radioactivity above control levels. The specificity of the PBR response was determined using a blocking dose of nonradioactive PK11195 given 30 min prior to radiotracer injection. Static planar images of the thorax of nonradioactive PK11195 pretreated animals showed a significantly lower level of radiotracer accumulation in control and in LPS-treated animals ( p < .05). These data show that LPS induces specific increases in PBR ligand binding in the lungs. We also used in vivo small-animal PET studies to demonstrate increased [11C]-( R)-PK11195 accumulation in the lungs of LPS-treated mice. This study suggests that measuring PBR expression using in vivo imaging techniques may be a useful biomarker to image lung inflammation.

Ex Vivo Generation of CD34+ Cells From CD34− Hematopoietic Cells

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4053-4059 ◽  
Author(s):  
Yoshihiko Nakamura ◽  
Kiyoshi Ando ◽  
Jamel Chargui ◽  
Hiroshi Kawada ◽  
Tadayuki Sato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Population ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Cd34 Cells

Abstract The human Lin−CD34− cell population contains a newly defined class of hematopoietic stem cells that reconstitute hematopoiesis in xenogeneic transplantation systems. We therefore developed a culture condition in which these cells were maintained and then acquired CD34 expression and the ability to produce colony-forming cells (CFC) and SCID-repopulating cells (SRCs). A murine bone marrow stromal cell line, HESS-5, supports the survival and proliferation of Lin−CD34− cells in the presence of fetal calf serum and human cytokines thrombopoietin, Flk-2/Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin-3, and interleukin-6. Although Lin−CD34− cells do not initially form any hematopoietic colonies in methylcellulose, they do acquire the colony-forming ability during 7 days of culture, which coincides with their conversion to a CD34+ phenotype. From 2.2% to 12.1% of the cells became positive for CD34 after culture. The long-term multilineage repopulating ability of these cultured cells was also confirmed by transplantation into irradiated NOD/SCID mice. These results represent the first in vitro demonstration of the precursor of CD34+ cells in the human CD34− cell population. Furthermore, the in vitro system we reported here is expected to open the way to the precise characterization and ex vivo manipulation of Lin−CD34− hematopoietic stem cells.

Uptake of Protamine Sulphate Complexed Fluorescent Nano-Particles Is Defined by Cell Cycle Status in Primary Human CD34+ Cells: Use of a Multi-Color p27 kip1 Based Flow Cytometric Assay.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1363-1363
Author(s):  
Ryan Lahey ◽  
Jesper Bonde ◽  
Jan A. Nolta
Keyword(s):  
Cell Cycle ◽  
In Vivo Imaging ◽  
Ex Vivo ◽  
Nano Particles ◽  
Flow Cytometry Assay ◽  
Hematopoietic Stem ◽  
Protamine Sulphate ◽  
Flow Cytometric ◽  
Cd34 Cells

Abstract The use of iron based nano-particles for multi-modal imaging is gaining interest, since it allows high resolution non-invasive in vivo imaging of human hematopoietic homing and engraftment events in xenograft models. The uptake of ferridex nano-particles complexed to cationic protamine sulphate is believed to be non-specific through mechanisms like endocytosis, but this has not been well defined for hematopoietic stem cells (HSC). In defining ex vivo cultivation strategies for manipulation of human HSC, a key factor is the responsiveness of the most primitive cells to the in vitro conditions, with the aim of maintaining viability without inducing terminal differentiation. Here, we present a novel flow cytometry assay which assesses the earliest molecular responses to a defined clinically applicable ex vivo protocol, aimed at facilitating labeling of human stem/progenitor cells using protamine sulphate complexed nano-particles for subsequent in vivo imaging. We used intracellular staining for the cell cycle inhibitor p27kip1, which is present in the highest levels in non-cycling cells, as the primary flow cytometric marker in combination with CD34, CD133 and Alexa 488, 647 and 750 conjugated ferridex nano-particles and the membrane dye PKH26. An assay was developed to simultaneously assess the molecular events occurring in individual human cord blood Lin− or CD34+ cells while they were cultured for up to 72 hours in X-Vivo 15 serum free medium supplemented with Flt3, SCF and TPO on Retronectin (RN) coated plates with or without nano-particles. Co-expression of p27kip1, CD34 or CD133 in the cultured cells slowly decreases from 86.1% CD34+p27kip1 (T=0) to 76.7%+/−12.2% (T=72) and from 89.6% CD133+p27kip1+ (T=0) to 54.1%+/−10.4% (T=72). We suggest that this slow decrease represents cells dividing and potentially differentiating over the time course of the ex vivo cultivation period. Assessing uptake of fluorescent conjugated nano-particles over a 72 hr period showed that the uptake of particles in CD34+ and CD133+ cells declined significantly after the first 24 hrs., from 32.5+/−3.7% nano-positive CD34+ cells to 19.2+/−2.9% at 48 hours ex vivo with a more significant decline to only 8.3+/−3.7% nano positive CD34+ cells in the culture after 72 hours ex vivo. The same decline in uptake over time was observed in cultured human CB cells that were positive for CD133. PKH26 co-staining demonstrated that the majority of cells that undergo cell division within the first 24 hours of ex vivo culture are the most likely to uptake the nano-particles. In summary, using a multi color p27kip1 based flow-cytometry assay, we found that human Lin−, CD133+, and CD34+ cells uptake Fe-Pro in a fashion which is not entirely cell cycle independent as previously suggested. These data indicate that cell cycle or metabolic status may influence the ability of human hematopoietic stem and progenitor subsets to uptake the protamine sulphate-complexed nano-particles. These findings emphasize the need to carefully develop ex vivo conditions for nano-particle labeling of primary human stem cells in order to perform accurate in vivo imaging of the most primitive human hematopoietic stem and progenitor cells.

In Vivo Imaging to Monitor Trafficking and Engraftment of Human CD34+ Hematopoietic Stem and Progenitor Cells in Rhesus Monkeys

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3495-3495
Author(s):  
Alice F. Tarantal ◽  
C. Chang I. Lee ◽  
Cynthia A. Batchelder ◽  
Julie L. Sutcliffe ◽  
Simon R. Cherry
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
In Vivo Imaging ◽  
Rhesus Monkeys ◽  
Firefly Luciferase ◽  
Hematopoietic Stem ◽  
Cord Blood Cd34 ◽  
Over Time

Abstract Significant advancements have been made in the use of noninvasive imaging techniques which includes dramatic improvements in resolution and sensitivity, and imaging systems designed specifically for animals. The non-invasive nature of these techniques allows longitudinal assessments over time, and the evaluation of protocols that could be applied in a human clinical setting. Imaging is also of great utility in situations when it is unclear where to focus the analyses, such as the challenges presented by systemic administration of transplanted cells, which can traffic to different anatomical sites. Our prior studies have shown that we can monitor gene expression in fetal and infant monkeys using reporter genes, microPET, and optical imaging. We have also shown that human mobilized peripheral blood stem cells (hPBSC) from adult male donors and male cord blood CD34+ hematopoietic stem cells (HSC) can readily engraft in fetal rhesus monkeys and persist over time. Other recent findings suggest that growth factor expansion of cord blood CD34+ HSC in vitro prior to transplant substantially increases the level of engraftment, with evidence of human hematopoietic cells in the rhesus host at levels significantly greater than the approximate 2% we previously reported. Our studies have also focused on new ways to use optical imaging to monitor transplanted hPBSC, cord blood HSC, and CD34+ precursors differentiated from human embryonic stem cells (hESC) that express firefly luciferase and a drug resistance gene, and the development of PET techniques for short-term tracking of transplanted cells. Transduced cells (1×104–20×106 cells/fetus) were transplanted intraperitoneally into fetal monkeys in the late first trimester under ultrasound guidance (N=27), then animals were monitored sonographically during gestation, and delivered by cesarean-section at term. At 1 week postnatal age and monthly thereafter animals were imaged for firefly luciferase expression (bioluminescence) following the intravenous injection of 100 mg/kg D-luciferin, and after the collection of blood and bone marrow for analysis. While evidence of the human Y chromosome (hSRY, hTSPY) was found in peripheral blood mononuclear cells and CD34+ immunoselected cells from marrow by qRT-PCR, the range of copies detected over time did not reflect the imaging findings. Foci of engrafted human cells were found to persist for ≥1 year, to date, in the rhesus host and in anatomical areas not previously identified (e.g., ribs, sternum, liver). In addition, 64Cu-PTSM radiolabeled hPBSC from the same donors were injected postnatally and cell trafficking monitored without evidence of toxicity or adverse effects. These in vivo imaging studies have provided unique insights into the fate of CD34+ cells post-transplantation, and indicate the importance of these techniques for cell transplant protocols.

Targeted Gene Modification In Hematopoietic Stem Cells: A Potential Treatment For Thalassemia and Sickle Cell Anemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 434-434
Author(s):  
Andreas Reik ◽  
Kai-Hsin Chang ◽  
Sandra Stehling-Sun ◽  
Yuanyue Zhou ◽  
Gary K Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Target Gene ◽  
Ex Vivo ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for <20% of cases, and (ii) the allogeneic nature of the transplant involves the significant risk of graft vs host disease (GvHD). Elevated levels of fetal γ-globin proteins observed in a subset of individuals carrying β-thal and SCD mutations ameliorate the clinical picture or prevent the development of disease complications. Thus, strategies for the selective and persistent upregulation of γ-globin represent an attractive therapeutic approach. Recent insights into the regulation of γ-globin transcription by a network of transcription factors and regulatory elements both inside and outside the β-globin locus have revealed a set of new molecular targets, the modulation of which is expected to elevate γ-globin levels for potential therapeutic intervention. To this end, we and others have established that designed zinc finger nucleases (ZFNs) transiently introduced into stem cells ex vivo provide a safe and efficient way to permanently ablate the expression of a specific target gene in hematopoietic stem cells (HSC) by introduction of mutations following target site cleavage and error-prone DNA repair. Here we report the development and comparison of different ZFNs that target various regulators of γ-globin gene transcription in human HSCs: Bcl11a, Klf1, and specific positions in the γ-globin promoters that result in hereditary persistence of fetal hemoglobin (HPFH). In all cases these target sites / transcription factors have previously been identified as crucial repressors of γ-globin expression in humans, as well as by in vitro and in vivo experiments using human erythroid cells and mouse models. ZFN pairs with very high genome editing activity in CD34+ HSCs were identified for all targeted sites (>75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (>108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (>16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.

Preclinical Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3859-3859
Author(s):  
Helen Fong ◽  
Goar Mosoyan ◽  
Ami Patel ◽  
Ronald Hoffman ◽  
Jay Tong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Shelf Life ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Hematopoietic Stem ◽  
Cell Product ◽  
Cd34 Cells

Abstract Platelet (PTL) transfusions are currently the most effective treatment for patients with thrombocytopenia. Demand for PTL transfusions has steadily increased in recent years, straining a PTL supply that is already limited due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. This dilemma has stimulated the search for alternative approaches for generating PTLs ex vivo from different sources of hematopoietic stem cells (HSCs). Although PTLs have been successfully generated in cultures initiated with primary human CD34+ cells and pluripotent stem cells, the generation of a clinically relevant PTL product ex vivo faces significant obstacles due to scalability, reproducibility and shelf life. We propose an alternative approach to overcome such obstacles by developing a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTL in vivo after transfusion into patients. Umbilical cord blood units (CBU) are FDA-approved, readily available sources for allogeneic HSC for transplantation in patients with various blood disorders. Our method utilizes a previously developed two-step culture system of megakaryopoiesis from CB CD34+ cells to generate an MK culture composed of defined MK populations: CD34+/CD41+/CD42b- MK precursors (MKP), immature CD34-/CD41+/CD42b- MK (iMK) and mature CD34-/CD41+/CD42b+ MK (mMK). While robust, the yield of MKs obtained in these cultures is restricted due to limited numbers of HSCs in CB. Our group has recently demonstrated that the numbers of CB CD34+ can be significantly expanded by epigenetic reprogramming following treatment with valproic acid (VPA). Here, we report the integration and optimization of HSC expansion with MK differentiation in order to generate a clinically relevant MK cell product. We tested 20 different culture conditions in which CD34+ cells were cultured for 5 to 8 days in the absence or presence of VPA in serum-free media with various cytokines to allow for HSC expansion. The resulting HSC pool is cultured for additional 4 to 7 days in MK differentiation/maturation media. The overall yield of CD41+ MKs obtained ranged from 8 to 33 MK per input CD34+ cell expanded in the presence of cytokines alone (n=10; mean 19.8 MK) and from 9 to 34 MK per input CD34+ cell expanded in the presence of cytokines plus VPA (n=10; mean 20.7 MK). Given that up to 2x106 CD34+ cells can be isolated from one CBU, it is anticipated that a culture yielding 28 or more MK per one CD34+ cell would generate over 56x106 MK or the equivalent of 7x105 CD41+ MK/kg/body weight for infusion into an 80 kg recipient. The culture conditions resulting in a yield of 28 or more MK per one CD34+ cell input are currently optimized to further maximize the fraction of MK generated which currently varies between 15-57% of culture. The predominant sub-population of MK resulted in these conditions consists of mMKs, regardless of VPA treatment. However, in the presence of VPA, the cultures contain a greater number of assayable CFU-MKs as compared to cytokines alone. Furthermore, preliminary studies suggest that transplantation of ex vivo generated MK leads to detectable human CD41+ cells into the BM and human PTL into the PB of NSG recipient mice. These results indicate that a MK cell product derived from CB HSCs expanded by VPA comprises not only mMK and iMK capable of immediate PTL release but also MKP and HPCs which are capable of sustained MK and PTL production. Another major advantage of a transfusion product composed of nucleated MKs is the possibility of storage by cryopreservation. Due to the fragility of mMK, we tested the cryopreservation of heterogeneous and purified MK cultures. Viability of cryopreserved MK cultures post-thaw was between 68.4-70% and no changes in the MK phenotype. Studies are ongoing to test the ex vivo and in vivo functionality of the cryopreserved MKs. In summary, starting with expanded CB HSC we created a cryopreservable cell product composed of different MK sub-populations within the MK hierarchy which is being developed into a clinically relevant therapy for treatment of thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

Large-Sacle ExVivo Generation of Human Red Blood Cells from Cord Blood CD34+ Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 817-817
Author(s):  
Yu Zhang ◽  
Chen Wang ◽  
Lan Wang ◽  
Jing Tian ◽  
Bin Shen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Transfusion ◽  
Blood Cells ◽  
Culture System ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Qpcr Analysis ◽  
Cd34 Cells ◽  
Human Rbcs

Abstract Blood transfusion is widely used for various clinical therapies. Ex vivo production of red blood cells (RBCs) in a large-scale from hematopoietic stem cells (HSCs) has been considered as a potential way to overcome the shortage of blood supply. Here, we report that functional human RBCs can be efficiently produced by using a bottle-turning device culture system from cord blood (CB) CD34+cells. A procedure of four-stage ex vivo expansion and differentiation was developed in a modified IMDM basal medium supplemented with transferrin, insulin, folic acid, fetal bovine serum, and some other nutrients with selected cytokine combinations that contained stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO) in stage I (days 0~5); SCF, FL, erythropoietin(EPO), interleukin 3 (IL-3), and GM-CSF in stage II (days 6~12); SCF, FL, IL-3, and EPO in stage III (days 13~18); SCF and EPO in stage IV (days 19~21). Enriched CD34+ cells were firstly cultured and expanded in 25-T flasks. After 5 day-culture, the cells were transferred to a 2-L bottle with 600 ml of medium in the bottle-turning device system. During the differentiation process, erythroid markers (CD71 and CD235a) and enucleation efficiency (LDS- percentage) of cultured cells were evaluated by flow cytometry. Erythroid progenitor cells were confirmed by clonogenic capacity by colony-forming unit (CFU) assay. Hemoglobin (HGB) content of the cells were determined quantitatively, and RT-qPCR analysis was performed to examine the expression of erythroid-specific genes and the status of proto-oncogenes. Furthermore, generated RBCs were CFSE-labeled and injected into irradiated NOD/SCID mice to monitor the viability and maturation in vivo. For stage I, the proliferation folds of CD34+ cells reached 20 ± 2.4 while CD34+ rate was maintained at 80% ± 4.3%. Subsequently, CFU assay on Day9 and Day12 showed that over 90% of the total colonies were erythroid burst-forming units (BFU-E) or erythroid colony-forming units (CFU-E), suggesting that these expanded cells were induced toward the erythroid lineage. For 21-day induction, approximate 2×108erythrocytes were produced from one CD34+cell with a purity of CD235a+ and CD71+ cells at 90% ± 6.2% and 54 % ± 7.2%, respectively. Furthermore, the results from flow cytometry of LDS-stained cells showed that 50% ± 5.7% of induced erythrocytes were enucleated. At various time points of the cell culture process, RT-qPCR analysis showed that expressions of erythroid-specific genes were normal and the proto-oncogenes (c-myc, c-myb, Bmi-1, k-ras, cyclinB, and HETRT) were not activated. From days 9 to 21, HGB contents of the cultured cells increased from 12.3 ± 1.5 pg/cell to 31.5 ± 2.4 pg/cell, which was similar to the contents of normal human RBCs (range: 27 - 33 pg/cell). In addition, the induced RBCs, after storing at 4℃ for more than 4 weeks, had normal HGB content, and showed normal expression of both CD235a and CD71. In mouse studies, the CD71+ marker on the 21-day cultured cells was diminished or undetectable in CFSE+ cells 3-day after transplantation. In contrast, LDS- cells among the CFSE+ population increased to 97.1% ± 2.3% 3-day post injection, indicating that the 21-day cultured RBCs could be further enucleated and matured in vivo. Taken together, we have established a pilot-scale culture system to produce functional human RBCs ex vivo. Considering that one blood transfusion unit contains 0.8×1012RBCs, the CD34+ cells from one CB unit (80 ml with 2×106 CD34+ cells) would generate 4×1014 RBCs, which are equivalent to 500 blood transfusion units in the clinical application. Disclosures Ren: Biopharmagen Corp: Employment. Jiang:Biopharmagen Corp: Consultancy.

An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3414-3421 ◽  
Author(s):  
Elen S. Rosler ◽  
John E. Brandt ◽  
John Chute ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Lymphoid Cells ◽  
Free Expansion ◽  
Hematopoietic Stem ◽  
Free System ◽  
Coculture System ◽  
Cd34 Cells

Abstract The marrow repopulating potential (MRP) of different sources of human hematopoietic stem cells (HSCs) was directly compared using an in vivo assay in which severe combined immunodeficient disease (SCID) mice were implanted with human fetal bones. HSCs from 2 human lymphocyte antigen (HLA)-mismatched donors were injected individually or simultaneously into the fetal bones of a 3rd distinct HLA type and donor and recipient myeloid and lymphoid cells were identified after 8 to 10 weeks. The study compared the MRP of umbilical cord blood (CB) and adult bone marrow (ABM) CD34+ cells as well as grafts of each type expanded ex vivo. Equal numbers of CB and ABM CD34+ cells injected individually demonstrated similar abilities to establish multilineage hematopoiesis. However, when CB and ABM cells were transplanted simultaneously, the engraftment of CB cells was markedly superior to ABM. CB and ABM CD34+ cells were expanded ex vivo using either a porcine microvascular endothelial cell (PMVEC)-based coculture system or a stroma-free expansion system. Primary CB CD34+ cells or CD34+ cells expanded in either culture system demonstrated a similar ability to engraft. However, the MRP of expanded grafts simultaneously injected with primary CB cells was uniformly inferior to primary CB cells. CD34+ cell grafts expanded in the stroma-free system, furthermore, outcompeted CD34+ cells expanded using the PMVEC coculture system. The triple HLA-mismatched SCID-hu model represents a novel in vivo stem cell assay system that permits the direct demonstration of the functional consequences of ex vivo HSC expansion and ontogeny-related differences in HSCs.

scholarly journals In Vivo Optical Reporter-Gene-Based Imaging of Macrophage Infiltration of DNCB-Induced Atopic Dermatitis

2020 ◽  
Vol 21 (17) ◽  
pp. 6205
Author(s):  
Sang Bong Lee ◽  
Hyeonsoo Park ◽  
Jae-Eon Lee ◽  
Kil-Soo Kim ◽  
Yong Hyun Jeon
Keyword(s):  
Atopic Dermatitis ◽  
Reporter Gene ◽  
In Vivo Imaging ◽  
Ex Vivo ◽  
Firefly Luciferase ◽  
Skin Lesions ◽  
Macrophage Infiltration ◽  
Living Organisms ◽  
Immunocompetent Mice

This study was conducted to monitor the macrophage infiltration of atopic dermatitis (AD)-like skin lesions and to evaluate the effects of anti-AD therapeutic agents in immunocompetent mice via optical reporter-gene-based molecular imaging. The enhanced firefly luciferase (effluc)-expressing macrophage cell line (Raw264.7/effluc) was intravenously introduced into mice with 2,4-dinitrochlorobenzene (DNCB)-induced AD, followed by bioluminescent imaging (BLI). After in vivo imaging, AD-like skin lesions were excised, and ex vivo imaging and Western blotting were conducted to determine the presence of infused macrophages. Finally, the therapeutic effect of dexamethasone (DEX), an AD-modulating agent, was evaluated via macrophage tracking. In vivo imaging with BLI revealed the migration of the reporter macrophages to DNCB-induced AD-like skin lesions on day 1 post-transfer. The greatest recruitment was observed on day 3, and a decline in BLI signal was observed on day 14. Notably, in vivo BLI clearly showed the inhibition of the reporter macrophage infiltration of DNCB-induced AD-like skin lesions by DEX, which was consistent with the reduced AD symptoms observed in DEX-treated mice. We successfully visualized the macrophage migration to DNCB-induced AD-like skin lesions, proving the feasibility of macrophage imaging for evaluating AD-regulating drugs in living organisms.

scholarly journals α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 558
Author(s):  
Laura Rodriguez ◽  
Pascale Duchez ◽  
Nicolas Touya ◽  
Christelle Debeissat ◽  
Amélie V. Guitart ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oxidative Phosphorylation ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Alpha Tocopherol ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
G0 Phase ◽  
The Impact

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect—the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Sign in / Sign up

Export Citation Format

Share Document