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.

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.

Download Full-text

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

Blood ◽

10.1182/blood.v106.11.3031.3031 ◽

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.

Download Full-text

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 ◽

10.1182/blood.v106.11.1363.1363 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood.v126.23.1899.1899 ◽

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.

Download Full-text

A Stemness Screen Reveals C3ORF54/INKA1 As a Gate-Keeper of Human Stem Cell Latency

Blood ◽

10.1182/blood-2018-99-115065 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 325-325

Author(s):

Kerstin B. Kaufmann ◽

Laura Garcia Prat ◽

Shin-Ichiro Takayanagi ◽

Jessica McLeod ◽

Olga I. Gan ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Fold Increase ◽

Steady Increase ◽

Post Transplantation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC. Our previous work on LSC subsets from >80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen. Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP. Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation. Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency. In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac. In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. Disclosures Takayanagi: Kyowa Hakko Kirin Co., Ltd.: Employment.

Download Full-text

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

Blood ◽

10.1182/blood.v95.9.2813.009k20_2813_2820 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2813-2820 ◽

Cited By ~ 215

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.

Download Full-text

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

Blood ◽

10.1182/blood.v94.7.2271.419k41_2271_2286 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2271-2286 ◽

Cited By ~ 86

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.

Download Full-text

Ex Vivo Modeling of Hematopoietic Stem Cell Homing to the Fetal Liver

10.1007/7651_2020_293 ◽

2020 ◽

Author(s):

Amina Mohammadalipour ◽

Miguel F. Diaz ◽

Sumedha Pareek ◽

Pamela L. Wenzel

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Ex Vivo ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Cell Homing ◽

Stem Cell Homing

Download Full-text

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

Blood ◽

10.1182/blood.v112.11.5393.5393 ◽

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.

Download Full-text

Targeting CD96 For Antibody Based Elimination Of Leukemic Stem Cells In AML: A New Strategy In Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.3972.3972 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3972-3972 ◽

Cited By ~ 2

Author(s):

Matthias Staudinger ◽

Christian Kellner ◽

Matthias Peipp ◽

Natalie Schub ◽

Andreas Humpe ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Target Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Although the mortality of autologous stem cell transplantation in contrast to allogeneic is low, in AML patients the lack of immune surveillance as well as contamination of the transplant with residual leukemic stem cells (LSC) limits its use. Therefore, elimination of LSC by targeted therapy may represent a promising therapeutic approach. Recently, CD96 was identified as marker antigen on AML-LSC (Hosen et al., PNAS 104: 11008, 2007). Here, by addressing CD96 with magnetic cell sorting (MACS) or using antibody dependent cellular cytotoxicity (ADCC), new strategies for engineering autologous stem cell grafts or for in vivo targeting of residual AML stem cells are presented. To evaluate the efficacy of depletion of LSC by MACS technology, grafts containing hematopoietic stem cells were spiked with CD96 positive AML cells. Using biotinylated CD96 antibody TH111 raised in our laboratory in combination with anti-biotin-micro beads (Miltenyi Biotech, Bergisch Gladbach, Germany) up to a 1000-fold depletion of targeted cells was achieved. The viability, cell count and the potential of hematopoietic progenitor cells (HPC) to proliferate and differentiate were not affected by this procedure as documented by flow cytometry and colony forming assays. As residual LSC residing within the patient may also account for AML relapse after high-dose chemotherapy and subsequent SCT, eradication of AML stem cells in vivo is desirable. To target CD96+ AML-LSC by ADCC, chimeric antibodies containing wild type or affinity maturated variable regions in combination with an optimized human IgG1Fc were generated by recombinant DNA technologies. Both recombinant antibodies were expressed in Hek 293 cells enriched to homogeneity by affinity chromatography and analyzed for their functional properties. As shown by flow cytometry, the antigen binding affinity of the maturated antibody was enhanced (EC50 0.6 μg/ml vs. 2 μg/ml). Moreover, as analyzed in standard ADCC assays, NK cell mediated lytic properties against CD96-positive target cells were elevated (maximum lysis: 52%) using the affinity maturated chimeric CD96 antibody (EC50: 0.02 μg/ml vs. 0.15 μg/ml). Thus, this CD96 purging strategy avoids unwanted transplantation of AML-LSC and may help to revitalize autologous stem cell transplantation in this indication. Although, specific side effects by CD96 application will have to be considered, this may allow for an additional therapeutic avenue to eliminate in vivo residual AML-LSC in autologous as well as in allogeneic situations. Disclosures: No relevant conflicts of interest to declare.

Download Full-text