A Potential New Application of Mobilization/Leukapheresis for Enrichment of Peripheral Blood in Circulating Non-Hematopoietic CXCR4+CD45− Tissue-Committed Stem Cells (TCSC) for Organ/Tissue Regeneration.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 151-151 ◽  
Author(s):  
Magda Kucia ◽  
Ryan Reca ◽  
Marcin Wysoczynski ◽  
Jolanta Gozdzik ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tissue Regeneration ◽  
Peripheral Blood ◽  
Organ Injury ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Heart Infarct ◽  
Partial Body ◽  
Organ Tissue

Abstract During mobilization hematopoietic stem cells (HSC) egress from the bone marrow (BM) into peripheral blood (PB) where they temporarily circulate and can be collected by leukapheresis. However, recently we demonstrated that BM, in addition to HSC, contains heterogeneous populations of CXCR4+ tissue-committed stem cells (TCSC) and we contend that the contribution of these cells to organ/tissue regeneration after transplantation of BM cells has been misinterpreted as evidence for “plasticity” or “trans-dedifferentiation” of HSC (Leukemia2004:18;29–40). To determine whether TCSC could also be mobilized into PB we i) evaluated the presence of these cells in the PB of G-CSF-mobilized patients (n=11), ii) attempted to increase mobilization of the TCSC in a murine model by combining G-CSF with T140 (a CXCR4 antagonist) or SB290157 (the C3a complement fragment receptor antagonist which we have found to desensitize the responsiveness of HSC to SDF-1), and iii) tested the hypothesis that TCSC could also be mobilized into PB during stress related to tissue/organ injury, e.g., heart infarct, stroke, partial body irradiation. The presence of mobilized/circulating TCSC in PB was evaluated by i) real-time RT-PCR, ii) immunohistochemical staining for TCSC markers and iii) demonstrating the potential of mobilized TCSC to grow neurospheres or to form myotubes in vitro. We present evidence for the first time that i) G-CSF efficiently enhances the release into PB not only of HSC but also of TCSC expressing markers for early skeletal muscle, myocardium, neural tissue, pancreas and liver, ii) a combination of G-CSF with T140 or SB290157 is 25–30 times more effective in selectively mobilizing TCSC compared to G-CSF alone, and iii) TCSC are also mobilized to PB during stress related to heart infarct, stroke or partial body irradiation. Furthermore, we observed that TCSC, like HSC, express CXCR4 and Sca-1 but do not radioprotect lethally irradiated mice and, unlike HSC, are CD45-negative. Based on these findings we postulate that mobilization/leukapheresis procedures may find a new application for obtaining TCSC for use in tissue/organ regeneration. We are currently testing this hypothesis in murine models.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

First Results of a Phase-II Study with the New CXCR4 Antagonist POL6326 to Mobilize Hematopoietic Stem Cells (HSC) In Multiple Myeloma (MM)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 824-824 ◽  
Author(s):  
Stefan Schmitt ◽  
Niels Weinhold ◽  
Klaus Dembowsky ◽  
Kai Neben ◽  
Mathias Witzens-Harig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dose Escalation ◽  
Peripheral Blood ◽  
Tumour Cells ◽  
Tolerability Profile ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Minimum Number ◽  
Cell Mobilization

Abstract Abstract 824 In multiple myeloma (MM), the second most common hematological malignancy, high-dose therapy followed by autologous CD34+ stem cell transplantation (ABSCT) is therapy of choice for younger patients. Standard treatment to mobilize hematopoietic stem cells (HSC) is either G-CSF alone or combined with chemotherapy. In the last years the antagonism of the CXCR4 receptor has been identified as a potent mechanism of HSC release from the bone marrow compartment. This mobilization by CXCR4 antagonists is more direct and more rapid than by G-CSF given over 4 to 6 days, and the combination of G-CSF plus a CXCR4 antagonist is superior to G-CSF alone. Furthermore, HSC mobilized using G-CSF and a CXCR4 antagonist have been shown to result in a rapid and sustained engraftment post-transplantation. POL6326 is a novel, potent and selective CXCR4 antagonist based on the PEM (Protein Epitope Mimetics) technology for intravenous application. In a previous Phase I study in 52 healthy volunteers POL6326 has been demonstrated to effectively mobilize CD34+ stem cells and was very well tolerated. The goal of the Phase II study in newly diagnosed myeloma patients reported here was to test to what extent POL6326 can be used as monotherapy, infused over 1 or 2h, to mobilize sufficient HSC for subsequent autologous transplantation. In our study the minimum number of HSC required for ABSCT was 2 × 106 CD34+ cells/kg body weight (BW). All patients also received CAD/G-CSF (cyclophosphamide, doxorubicin and dexamethasone) about 10 days after POL6326 and leukapheresis. The second goal was to determine tumour cell mobilization by polymerase chain-reaction after POL6326 or CAD/G-CSF respectively. Here we report the first data of this ongoing study. In all doses tested, up to 1200 μg/kg BW over 2h, POL6326 was safe and very well tolerated. During a total number of 38 infusions the only minor adverse event possibly related to study drug was a discrete pruritus of CTC grade 1 at the infusion site during POL6326 administration on two consecutive days. It resolved spontaneously and did not require any medication. This excellent safety and tolerability profile warrants further dose escalation in the ongoing trial. After first diagnosis, patients received 3 cycles of induction chemotherapy with bortezomib (or thalidomide respectively in one single subject) plus doxorubicin and dexamethasone. 3 weeks after the last injection of induction treatment HSC were mobilized with POL6326 given as a 1 or 2h infusion on up to 4 consecutive days according to an intra-individual dose escalation scheme (from 600 to 1200 μg/kg) with consecutive leukapheresis 30 min after end of infusion. In all patients (n=16) we observed an elevation of leukocytes and HSC compared to baseline. In 66% of patients sufficient stem cells were mobilized with POL6326 for ABSCT (with a mean of 2.5 × 106 CD34+ cells/kg BW). In 75 % of these subjects 2 leukapheresis cycles were sufficient to reach the minimum number of HSC. In all patients undergoing ABSCT with HSC mobilized by POL6326, the engraftment was successful. The time to reconstitution ranged from 10 to 19 days (mean 14 days) and was comparable to ABSCT with CAD/G-CSF-mobilized HSC (historical mean of 14 days and a range from 6 to 31 days). Five patients were evaluated for the presence of tumour cells in peripheral blood before mobilization and in leukapheresis products after treatment with POL6326 and CAD/G-CSF. In 2 of these patients we could not detect any tumour cells, neither in all leukapheresis products nor in all peripheral blood samples. In 2 patients a minor contamination with less than 0.0001% of tumour cells was detected both in the peripheral blood before POL6326 and in the leukapheresis products. Finally, 1 patient showed a similarly minor (<0.0001%) percentage of tumour cells only in the apheresis product after POL6326 treatment. However, a significant mobilization of tumour cells (0.001%) was detected after CAD plus G-CSF in this patient. Due to the excellent safety and tolerability profile observed for POL6326 in this study the next steps include further dose escalation. These initial data with low doses of POL6326 indicate that this novel CXCR4 antagonist holds the promise to be eventually used as a stand alone therapy not requiring the application of G-CSF. The preliminary findings of the absence or very low counts of tumour cells in the leukapheresis product generated with POL6326 warrant further investigation. Disclosures: Off Label Use: POL6326 as new CXCR4 antagonist for experimental CD34+ hematopoietic stem cell mobilization. Dembowsky:Polyphor Ltd: Employment. Braun:Cytonet Heidelberg GmbH: Employment. Ludin:Polyphor Ltd: Employment.

A Novel View of Bone Marrow As a “stem Cell sensor” of Tissue/Organ Damage -Evidence That in Vivo Exposure to the Neurotoxin Kainic Acid (KA) Induces Proliferation and Neural Specification of Developmentally Early Stem Cells Directly in Bone Marrow Before They Are Mobilized Into Peripheral Blood

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1192-1192
Author(s):  
Katarzyna Grymula ◽  
Maciej Tarnowski ◽  
Malwina Suszynska ◽  
Katarzyna Piotrowska ◽  
Sylwia Borkowska ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Brain Damage ◽  
Peripheral Blood ◽  
Organ Damage ◽  
Organ Injury ◽  
Neural Lineage ◽  
Heart Infarct

Abstract Abstract 1192 Background. It is well known that various stem cells become mobilized into peripheral blood (PB) in response to tissue/organ injuries (e.g., heart infarct, stroke, or bleeding); however, the data on the immediate response of stem cells in BM during organ injuries are somewhat limited. We and others have demonstrated the presence of developmentally early stem cells in BM that we have named very small embryonic-like stem cells (VSELs). These Oct-4+SSEA-1+Sca-1+Lin–CD45– cells are kept quiescent in BM in the G0 phase of the cell cycle by erasure of the somatic imprint in the differentially methylated regions (DMRs) of some crucial paternally imprinted genes, (Igf2-H19, RasGRF1, and p57Kip2) that regulate proliferation of embryonic stem cells (Leukemia 2009;23:2042). These cells are mobilized into peripheral blood, for example, during heart infarct (J Am Coll Cardiol 2009;6:1–9.), stroke (Stroke 2009;40:1237–44.), or skin burns (Stem Cell Rev. 2012;8:184–94.). Hypothesis. We hypothesized that this population of BM-residing, small, quiescent, pluripotent cells should be able to respond to organ injury induced by a known neurotoxin, kainic acid (KA), in a brain damage model. We hypothesized that these quiescent cells would began to proliferate, expand, and become specified into the neural lineage. Experimental strategies. C57Bl6 mice were injected with increasing doses of KA and at various time intervals mice were sacrificed to harvest BM, PB samples, and brains for analysis. Brain damage was confirmed by histological analysis. The number of Sca-1+Lin–CD45– VSELs and Sca-1+Lin–CD45+ HSPCs was evaluated in BM and PB by FACS. The cell cycle status of VSELs and HSPCs was evaluated by FACS in cells isolated from mice that received bromodeoxyuridine (BrdU) after KA injection. By employing RQ-PCR, we also measured the expression of genes that regulate stem cell pluripotency (Oct-4, Nanog, Sox2, and Rex1) and regulate neuronal development (Nestin, βIII-tubulin, Olig1, Olig2, and GFAP). The expression of these genes was subsequently confirmed in sorted cells by immunohistochemical staining. The numbers of clonogenic CFU-GM and BFU-E progenitors residing in BM and circulating in PB were tested in methylcellulose cultures. Results. We found that 12 hrs after administration of KA (25 mg/kg bw) quiescent VSELs residing in BM enter the cell cycle: ∼2 ± 1% for control vs. 37 ± 6% for KA-treated cells. Interestingly, at the same time we did not observe significant changes in the proliferation rate of HSPCs (15±5% for control vs. 17±4% for KA-treated cells). The elevated number of VSELs in the cell cycle remained detectable for a few days and returned to control values (∼2%) after 1 week after KA administration. Furthermore, an increase in the number of cycling VSELs correlated with an increase in expression of pluripotent markers, according to RQ-PCR analysis. In parallel, 48 hrs after KA administration we observed the release from BM into PB of Sca-1+Lin–CD45–VSELs highly enriched for mRNAs characteristic of neural differentiation. Interestingly, while we observed a significant increase in VSEL number in BM and PB after KA-induced brain damage, no significant changes were observed for both BM-residing and circulating HSPCs. Conclusions. For the first time, we provide evidence that the compartment of developmentally early stem cells residing in BM responds robustly to brain damage induced by a neurotoxin. This effect seems to be specific for VSELs, as no significant changes were observed for HSPCs. The kinetics of changes in BM revealed that BM VSELs enter the cell cycle and, after they become specified into the neural lineage, egress from BM and enter the PB. Thus, our data provide novel evidence that developmentally early stem cells in BM “sense” the damage to brain tissue and respond to this type of organ injury. In parallel, we are studying the specificity of the response of BM-residing VSELs and HSPCs to other types of organ damage, such as heart infarct and acute limb ischemia. Disclosures: Ratajczak: Neostem Inc: Member of SAB Other.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

Disruption of Tet2 Leads to Enhanced Self-Renewal and Competitive Repopulating Capacity of Fetal Liver Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2331-2331
Author(s):  
Hiroyoshi Kunimoto ◽  
Yumi Fukuchi ◽  
Masatoshi Sakurai ◽  
Ken Sadahira ◽  
Yasuo Ikeda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Gene Trap ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Serial Transplantation

Abstract Abstract 2331 TET2 (ten-eleven-translocation 2) gene has been reported to be frequently mutated in various human myeloid malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, acute myeloid leukemia, and chronic myelomonocytic leukemia. These observations suggest critical roles of TET2 dysfunction in molecular pathogenesis of myeloid malignancies. Recent studies using conditional knockout mouse model indicated that mouse Tet2 loss leads to clonal dominance of adult hematopoietic stem cells (HSCs) in competitive repopulation assay. However, self-renewal capacity of adult HSCs has never been addressed precisely by serial transplantation assay. In addition, the effect of Tet2 loss on hematopoietic stem/ progenitor cells was examined only in the BM, but not in the fetal livers (FLs). Since FL HSCs and adult HSCs differ in several aspects of their phenotypes and functions, we speculated that Tet2 might be involved differently in the regulation of FL and adult hematopoiesis. To address this issue, we analyzed E14.5 FL cells from Tet2 gene-trap (Tet2gt) mice. In these mice, gene trap-cassette was inserted into the second intron, just before the first coding exon. RT-PCR analysis showed that over 99% of Tet2 mRNAs from endogenous promoter were trapped by the gene-trap cassette in Tet2gt/gt mice, showing that Tet2gt allele can be considered as a null allele. Initial analysis showed that Tet2gt/gt embryos developed normally, but most Tet2gt/gt mice were perinatally lethal. Total numbers of FL cells and the numbers of committed progenitors in FLs as revealed by colony assays were not significantly different between each genotype. Interestingly, Tet2gt/gt embryos displayed significant increase in lineage (Lin)(-)Sca-1(+)c-Kit(+)(LSK) fraction compared to wild type (WT) (Tet2+/+) littermate (2.42±0.66% vs. 1.17±0.18%, p=0.02). In addition, common myeloid progenitor (CMP) fraction (IL7Rα(-), Lin(-), Sca-1(-), C-Kit(+), CD34(+), FcgRII/ III(low)) was significantly increased in Tet2gt/gt FLs compared to WT (9.04±1.09% vs. 6.26±0.53%, p=0.008). In serial transplantation assays, donor cells derived from Tet2+/gt and Tet2gt/gt FLs showed significantly higher peripheral blood chimerism in secondary and tertiary recipient mice as compared to that of WT cells, showing that disruption of Tet2 leads to the enhanced self-renewal capacity of FL HSCs. Moreover, donor-derived HSC fraction (CD34−LSK cells) was significantly expanded in the recipients of Tet2gt/gt FL cells, suggesting that increased self-renewal capacity is cell intrinsic to Tet2gt/gt HSCs. We have also examined differentiation of Tet2-mutant FL cells in the recipients' peripheral blood, and found that Tet2gt/gt cells displayed impaired differentiation to Gr-1(+)CD11b(+) mature granulocytes (WT vs. Tet2gt/gt = 5.02±1.35% vs. 11.5±3.09% in the primary recipients) and slight, but significant increase of B cells. Liquid culture of FL cells with cocktails of cytokines in vitro demonstrated that Tet2gt/gt FL cells retained higher percentage and number of LSK, Lin- and c-Kit+ cells after the culture for 7-days compared to WT cells, showing enhanced resistance of Tet2gt/gt cells to differentiative stimuli in in vitro culture. It is of note that Tet2+/gt mice showed a significant increase in hematopoietic stem/progenitor fraction (LSK) in the BM compared to wild type littermate (0.48±0.11% vs. 0.32±0.04%, p=0.04). However, they presented no signs of extramedullary hematopoiesis such as splenomegaly and expansion of LSK cells in spleens during an observation up to 35-weeks. Taken together, we demonstrate that Tet2 critically regulates self-renewal and long-term repopulating capacity of FL HSCs and has pleiotropic functions in myeloid and lymphoid differentiation. These data strongly indicate that Tet2 is an essential regulator of BM and FL hematopoiesis. In addition, enhanced HSC self-renewal, expansion of HPC and myeloid progenitors and perturbed myeloid differentiation induced by TET2 ablation likely to set molecular basis for myeloid transformation, which explains high incidence of loss-of-function mutations of TET2 in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

Ex Vivo Expanded Megakaryocyte Progenitors Generated From Human Mobilized Peripheral Blood CD34+ Stem Cells Can Be Cryopreserved and Transplanted Into NSG Mice to Generate Functional Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 482-482
Author(s):  
Holger Karsunky ◽  
Robert J. Tressler ◽  
Joy Chananukul
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Platelet Transfusion ◽  
Ex Vivo ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Mobilized Peripheral Blood ◽  
Megakaryocyte Progenitors

Abstract Abstract 482 Thrombocytopenia is a common side effect of high-dose chemotherapy that can compromise cancer treatment by requiring treatment delay and/or dose reduction for the patient. Platelet transfusion is typically given to prevent severe hemorrhage. However, several factors including acquisition, banking, and associated risks of bacterial infections and alloimmunization are hampering reliance on platelet transfusion. Growth factors are also used to stimulate proliferation and differentiation of megakaryocytes to increase platelet production, but in severely myelosuppressed patients these have only had modest benefit. The limitations of these two modalities for the treatment of chemotherapy-induced thrombocytopenia indicates that additional treatment approaches are needed. We have developed a novel approach to reconstitute megakaryocytes and platelets in thrombocytopenic patients which is presented here. We have identified a scalable culture system using serum-free medium and a defined cytokine cocktail free of animal products to expand CD34+ hematopoietic stem cells from G-CSF mobilized peripheral blood donors in vitro and direct their development to the megakaryocyte lineage to yield committed human megakaryocyte progenitors (MKPs). These MKPs can be readily cryopreserved while retaining their capacity to generate CFU-MK and platelets in vitro. When infused into NSG mice, ex vivo expanded MKP generate clinically relevant platelet levels of platelets in blood within a few days with sustained platelet levels for several weeks. The platelets generated from MKP in vivo are also functional as assessed by CD62P expression in responses to ADP stimulation in vitro. Our results present a compelling approach for the development of off-the-shelf storable MKPs for the treatment of thrombocytopenia. Disclosures: Karsunky: Cellerant Therapeutics Inc.: Employment, Patents & Royalties. Tressler:Cellerant Therapeutics, Inc.: Employment, Equity Ownership. Chananukul:Cellerant Therapeutics Inc.: Employment, Patents & Royalties.

Novel Double Labeled Mixed Lymphocyte in Vitro Assay to Predict the Dominant Graft in 2 Unit UCB Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4699-4699
Author(s):  
Shicheng Yang ◽  
Xiao Huang ◽  
Hongyan Lu ◽  
Amandeep Salhotra ◽  
Alexander Wendling ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
In Vitro Assay ◽  
Proliferation Index ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Ifn Γ

Abstract Abstract 4699 Introduction: Umbilical cord blood cells (UCB) from allogeneic donors have been established as an alternative source for HSC transplantation in patients who lack suitably HLA matched bone marrow or peripheral blood stem cells from adult donors. Transplantation using 2 unit UCB has been shown to compensate the low engraftment and slow hematopoietic recovery resulting from 1 unit UCB transplantation in full stature adult patients. At present, there are no unit specific factors that reliably predicts for the “winning unit” in 2 unit UCB transplantation, e.g. cell viability, number of infused total nucleated cells, CD34+ or CD3+ cells, sex mismatch, ABO blood group, and degree of HLA mismatch. In vivo mouse models suggest that CD34 negative subsets play an important role. Among CD34 negative subsets, CD8 T subset accounts for approximately 34.0+/−23.3% of T lymphocytes from UCB. In bone marrow transplantation CD8 T cells have been found to facilitate donor hematopoietic cell engraftment. Moreover, it has been reported that 1 dominant unit coincides with a specific CD8 T cell response against the non-engrafted unit which was not observed from CD4 or NK cells. Methods: In this study, we used volunteer donated UCB research units (kindly provided by P. Rubinstein, MD, New York Blood Center). Mononuclear cells (MNC) were purified by Ficoll gradient centrifugation, and CD3 T cells were isolated with CD3 MicroBeads (Miltenyi Biotec; autoMACS). The purified CD3 (confirmed by FACS >95% purity) cells were labeled with CFSE and DDAO-SE. After labeling, the cells from two different donors were mixed in 96-well U-bottom plates for continued culture in 37 °C 5% CO2. The expansion from each labeled donor cells was evaluated using flow cytometry; the dead cells were gated out using propidium iodide, and the data was analyzed using FlowJo software. For proper T cells activation, we also compared different activation conditions using i.) anti-CD3/CD28 Beads, ii.) anti-CD3 antibody plus anti-CD28 antibody, and iii.) cytokine IL-2. The schematic illustration of methods is shown in Figure 1. Results and discussion: We noted that T cells from UCB are primarily at naïve stage as determined by CD45RA (93.8 +/− 7.11%) and CCR7 (84.9 +/− 12.0%) expression. We also determined the optimal activation condition using a modified mixed lymphocyte reaction from 2 UCB units. Four days after incubation, the proliferation from 2 units labeled with CFSE and DDAO-SE could be reproducibly distinguished using FL1 channel for CFSE and FL4 channel for DDAO-SE (Figure 1). The optimal concentration for labeling using CFSE (1 mM) and DDAO (1 μM or 3 mM) was determined by titration. To avoid cell toxicity resulting from CFSE and DDAO-SE labeling, as well as self-crossing from each donor using two dyes, we examined additional mixed lymphocyte analyses in which each donor was labeled with CFSE or DDAO-SE respectively and vice versa. As shown in Figure 1, we found consistently that the predicated dominant unit accounted for the majority of culture (73.2% stained with DDAO; 63.5% stained with CFSE) after 4 days co-culture. The dominance was not correlated with cell proliferation indicated by the proliferation index (1.12 for dominant and 1.48 for another unit). After confirmation of this in vitro assay, further studies were conducted to evaluate the IFN-γ release of 2 UCB units in this optimized mixed lymphocyte assay in the condition using cytokine IL-2. Interestingly, we could only detect IFN-γ by intracellular staining in one unit when co-culture was set-up using CD3 T cells from each unit; the expression of IFN-γ was not detected when we used CD3 T cells from 1 unit. The correlation between dominance and the expression of IFN-γ is currently under investigation. Conclusion: UCB Transplantation is an important alternative for patients lacking bone marrow or peripheral blood stem cell donors. With the establishment of this novel modified mixed lymphocyte in vitro assay for prediction of the “winning” immune dominant unit, routine analyses can be performed to guide unit selection. Further interventions can be exploited to preferentially treat the expected dominant unit with glycosylation, cytokines, prostaglandins, or C3a compliments to further enhance hematopoietic stem cells trafficking and engraftment to the marrow. Disclosures: No relevant conflicts of interest to declare.

More Evidence That the Phenomenon of “Plasticity” of Hematopoietic Stem Cells (HSC) and Potential Involvement of BM-Derived Cells in Organ Regeneration Could Be Explained by the Presence of a Heterogenous Population of Tissue Committed Stem Cells (TCSC) Residing/Hiding out in Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4173-4173
Author(s):  
Magda Kucia ◽  
Marcin Wysoczynski ◽  
Ryan Reca ◽  
Marcin Majka ◽  
Janina Ratajczak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mononuclear Cells ◽  
Immunohistochemical Analysis ◽  
Organ Injury ◽  
Older Individuals ◽  
Hematopoietic Stem ◽  
Organ Regeneration ◽  
Partial Body ◽  
Heterogenous Population

Abstract Recently we identified a mobile, SDF-1 responsive population of CXCR4+ bone marrow mononuclear cells that express mRNA for various markers of early tissue committed stem cells (TCSC) in bone marrow (BM) by employing chemotactic isolation to an SDF-1 gradient combined with real time RT-PCR and immunohistochemical analysis (Leukemia2004:18;29-40). In the current work we report new data showing that murine and human TCSC (i) are highly mobile and respond robust to HGF and LIF gradients, in addition to an SDF-1 gradient, (ii) are released (mobilized) from BM into peripheral blood during organ injury (e.g., stroke, heart infarct, partial body irradiation), (iii) are highly enriched in mRNA for pluripotent stem cell transcription factors such as Oct-4, Rex-1 and Nanog, iv) reside in a population of BM-derived Sca-1+ (mice) and CD34+AC133+ (human) non-hematopoietic CD45 negative cells, v) are small (~7 mm in diameter), vi) contain supercoiled DNA, vii) rapidly adhere to/undergo emperipolesis in fibroblasts and thus are enriched in a fraction of BM-derived adherent cells, viii) their number is highest in BM from young (1–2 month-old) mice and decreases in 1-year-old animals, and similarly ix) these cells are significantly diminished in short living DBA/2J mice as compared to long living B6 mice. Thus, these findings further support our theory of the BM as a “hideout” of TCSC and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as trans-dedifferentiation/plasticity of HSC. Since we noticed that not only SDF-1 but also HGF and LIF are upregulated in damaged tissues (e.g., brain, heart or liver), we postulate that TCSC that express CXCR4, c-Met, and LIF-R after being mobilized from the BM into PB, may be subsequently chemoattracted to damaged organs where they play a role in tissue repair/regeneration. Moreover, our observation that the number of TCSC is the highest in BM of young animals and decreases with age and is significantly lower in short-living murine strains provides a novel insight into aging and may explain why the regeneration process becomes less effective in older individuals. Based on our observation, we conclude that BM-derived HSC are not plastic but BM in addition to HSC also contains heterogenous populations of TCSC.

scholarly journals Differential Expression of the Tetraspanin CD9 in Normal and Leukemic Stem Cells

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 312
Author(s):  
Rachid Lahlil ◽  
Maurice Scrofani ◽  
Anne Aries ◽  
Philippe Hénon ◽  
Bernard Drénou
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Myeloproliferative Neoplasms ◽  
Cellular Growth ◽  
Organ Injury ◽  
Hematopoietic Stem ◽  
Cell Therapies ◽  
Surface Receptors ◽  
Myeloid Neoplasms

CD9 plays a crucial role in cellular growth, mobility, and signal transduction, as well as in hematological malignancy. In myeloid neoplasms, CD9 is involved in the altered interactions between leukemic and stromal cells. However, apart from its role in CD34+ progenitors and myeloid and megakaryocytic differentiation, its function in normal and leukemic pluripotent cells has not yet been determined. Very small embryonic-like stem cells (VSELs) are promising pluripotent stem cells found in adult tissues that can be developed for safe and efficient regenerative medicine. VSELs express different surface receptors of the highest importance in cell functioning, including CD9, and can be effectively mobilized after organ injury or in leukemic patients. In the present study, we observed that CD9 is among the most expressed receptors in VSELs under steady-state conditions; however, once the VSELs are expanded, CD9+ VSELs decrease and are more apoptotic. CD9– VSELs had no proliferative improvement in vitro compared to those that were CD9+. Interestingly, the addition of SDF-1 induced CD9 expression on the surface of VSELs, as observed by flow cytometry, and improved their migration. In addition, we observed, in the phenotypically identical VSELs present in the peripheral blood of patients with myeloproliferative neoplasms, compared to healthy subjects, a significantly higher number of CD9+ cells. However, in their hematopoietic stem cell (HSC) counterparts, the expression remained comparable. These results indicate that, likewise, in progenitors and mature cells, CD9 may play an important function in normal and malignant VSELs. This could explain the refractoriness observed by some groups of expanded stem cells to repairing efficiently damaged tissue when used as a source in cell therapies. Understanding the function of the CD9 receptor in normal and malignant CD34+ and VSELs, along with its relationship with the CXCR4/SDF-1 pathway, will enable advances in the field of adult pluripotent cell usage in regenerative medicine and in their role in leukemia.

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