Differential Effects of a Novel Non-Peptidic Thrombopoietin Mimetic on Proliferation and Differentiation of Human CD34+ Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2888-2888 ◽  
Author(s):  
Juha Punnonen ◽  
Marcus O. Muench ◽  
Jeffrey R. Spencer
Keyword(s):  
Molecular Weight ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Aqueous Solubility ◽  
Similar Proportion ◽  
Low Molecular Weight ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Megakaryocyte Differentiation

Abstract Thrombopoietin (TPO) and TPO mimetics have been shown to be beneficial in the treatment of thrombocytopenia. Low molecular weight, orally available compounds offer the advantage of convenience and lack of immunogenicity when compared to protein-based drugs. STS-T4 is a novel low-molecular weight (<500 Da), non-peptidic TPO mimetic compound that is a 2nd generation agent in preclinical development with promising in vitro potency and aqueous solubility. We have studied the effects of STS-T4 on the growth and differentiation of primary human CD34+ progenitor cells. A dose-dependent proliferation of highly purified human CD34+ cells was observed with EC50 value of less than 1 μM. A bell-shaped dose-response curve was observed, which is consistent with previous reports on compounds stimulating homodimeric cytokine receptors. In addition, the phenotype of the cultured cells was analyzed by flow cytometry and monoclonal antibodies specific for markers of megakaryocyte differentiation. During a culture period of 10 days, 85% of the cells cultured in the presence of STS-T4 differentiated into CD41+ megakaryocytes in the absence of any other cytokines or mimetics. In addition, 30% of these CD41+ cells coexpressed CD42b as a marker of more mature cells. When compared to recombinant human TPO (rhTPO), a similar proportion of the cultured cells expressed CD41 and CD42b in response to STS-T4 and the levels of expression of these antigens on the surface of the megakaryocytes were similar, suggesting that the effects of STS-T4 and rhTPO on human megakaryocyte differentiation are comparable. However, the level of cell proliferation induced by STS-T4 in cultures of CD34+ cells was 50–60% of that induced by rhTPO, supporting the conclusion that the signals mediating proliferation and differentiation of CD34+ progenitor cells are differentially regulated. In addition to the activity profile, the physical properties of STS-T4 are desirable for further development. Aqueous solubility and gastrointestinal permeability are major contributors to oral absorption, and high solubility and permeability generally also reduce the risk of food effects. The aqueous solubility of STS-T4 was measured by a kinetic method with HPLC and visual detection and was determined to be >1 mM. These data suggest that further evaluation of safety and efficacy of STS-T4 for the treatment of thrombocytopenia is warranted. In addition, the results imply that CD34+ progenitor cells can differentiate into megakaryocytes expressing high levels of CD41 and CD42b in the absence of a full rhTPO-like proliferative response, which may support further investigation of new therapies for the treatment of thrombocytopenia that provide the benefit of megakaryocyte maturation while avoiding excessive expansion of hematopoietic stem cells.

Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3975-3975
Author(s):  
Szabolcs Fatrai ◽  
Djoke van Gosliga ◽  
Lina Han ◽  
Simon M. G. J. Daenen ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Signal Transduction Pathways ◽  
Monocytic Differentiation ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
P38 Pathway

Abstract Abstract 3975 Poster Board III-911 In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved. In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V. In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways. Disclosures: No relevant conflicts of interest to declare.

Abstract 42: In Vivo Differentiation of Epigenetically Reprogrammed Human Endothelial Progenitor Cells into Cardiomyocytes Enhances Functional and Anatomical Postinfarct Myocardial Repair

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Melissa A Thal ◽  
Prasanna Krishnamurthy ◽  
Alexander R Mackie ◽  
Eneda Hoxha ◽  
Erin Lambers ◽  
...  
Keyword(s):  
Heart Disease ◽  
Ischemic Heart Disease ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Ischemic Heart ◽  
Specific Gene ◽  
Myocardial Repair ◽  
Endothelial Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells

Currently, bone marrow derived endothelial progenitor cells (human CD34+ cells, EPC) are being used clinically to improve vascularization in patients with ischemic heart disease. While it is generally accepted that CD34+ cells predominantly work through a paracrine mechanism, there exists no convincing evidence that these cells trans-differentiate into functional cardiomyocytes (CMC). Since ischemic heart disease leads to substantial loss of CMC, improving cardiomyogenic plasticity of an existing autologous cell therapy is of obvious import. EPC and CMC both differentiate from a common mesodermal progenitor however; during EC-specific lineage differentiation, CMC specific genes are epigenetically silenced. We hypothesized that reprogramming of CD34+ cells using small molecules targeting key epigenetic repressive marks may recapitulate their cardiomyogenic potential. Human CD34+ EPCs were treated with inhibitors of histone deacetylases (valproic acid) for 24 hours followed by an additional 24 hours with the DNA methyltransferase inhibitor (5-Azacytidine). This forty-eight hour treatment led to the reactivation of pluripotency associated and CMC specific mRNA expression while EC specific gene expression was maintained. Intra-myocardial transplantation of a sub-therapeutic dose of reprogrammed CD34+ cells in an acute myocardial infarction mouse model showed significant improvement in LV function compared to the same number of control CD34+ cells that are therapeutically equivalent to no treatment at all. This was histologically supported by de novo CMC differentiation. In addition to increased cardiomyogenic plasticity, drug treatment also enhanced the inherent therapeutic capacity of the CD34+ cells as shown by reduced fibrosis, increased capillary density, increased proliferation, increased cell survival and increased secretion of angiogenic factors. Taken together, our results suggest that epigenetically reprogrammed CD34+ cells are “super-CD34+ cells” that have an enhanced paracrine effect, display a more plastic phenotype and improve post-infarct cardiac repair by both neo-cardiomyogenesis and neovascularization.

MKL1 Enhances Megakaryocytic Differentiation of Primary CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2218-2218
Author(s):  
Matthew J. Renda ◽  
James A. Troy ◽  
Ee-Chun Cheng ◽  
Lin Wang ◽  
Diane S. Krause
Keyword(s):  
Lentiviral Vectors ◽  
Absolute Number ◽  
High Concentration ◽  
Megakaryoblastic Leukemia ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Megakaryocyte Differentiation ◽  
In Cells

Abstract Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and young children, is characterized by a failure of megakaryocyte (MK) differentiation, bone marrow fibrosis, cytogenetic abnormalities, and a poor prognosis. We are particularly interested in AMKL that is associated with the translocation t(1;22)(p13;q13), which yields an in-frame fusion of RBM15 (OTT) and MKL1 (MAL) on chromosomes 1 and 22, respectively. The resultant fusion, RBM15-MKL1 is believed to include all of the functional domains of each component. In order to better understand the role of RBM15-MKL1 in AMKL, it is necessary to understand the roles of the constituent genes, RBM15 and MKL1, in hematopoiesis. We have studied the role of human MKL1 in megakaryopoiesis using primary human CD34+ cells purified from G-CSF mobilized PBMC from healthy donors (n=4). To optimize the CD34+ model, we tested the ability of TPO vs. TPO+SCF vs. TPO+SCF+IL–3 to induce megakaryocytopoiesis. TPO and TPO+SCF gave the highest percentages of MK (12% and 7%, respectively) on day 9. However, due to enhanced cell proliferation with TPO+SCF, the absolute number of MK was highest using this cytokine combination. To test the effect of MKL1 overexpression on megakaryopoiesis, we generated VSVG-pseudotyped lentiviral vectors containing human MKL1 and tested the effect of retronectin on viral transduction of CD34 cells. Surprisingly, retronectin decreased the level of transduction when compared to no retronectin (12% vs. 15% transduction respectively). We also found that polybrene enhanced transduction compared to lipofectamine 2000 (20% vs. 6% transduction, respectively). Using our optimized protocols, we examined the effect of MKL1 overexpression on megakaryocytopoiesis. One million CD34+ cells were thawed, infected the following two days with either empty lentivirus (pCCL) or lentivirus containing human MKL1 (pCCL-MKL), and cultured in TPO+SCF for 9 days. Since both lentiviral vectors included GFP driven by the PGK promoter, we measured the levels of CD41a, CD42d, and CD61 in GFP+ cells at day 9. In a representative experiment (of 4), CD41a levels increased in cells containing pCCL-MKL1 vs. pCCL (50% vs. 40%). Moreover, CD42d levels (22% vs. 7%) and CD61 levels (53% vs. 44%) were increased in cells containing pCCL-MKL1 virus when compared to cells containing pCCL virus. We also tested the ability of MKL1 to increase megakaryocyte differentiation using a semisolid Megacult assay from Stem Cell Technologies. CD34+ cells were cultured and infected as described above with either pCCL or pCCL-MKL1 virus. Two days post infection, GFP+ cells were FACS sorted and plated at two different concentrations in semisolid Megacult medium containing collagen, TPO, IL-6, and IL-3. Eleven days post plating, cells were stained for CD41/CD61. Cells infected with pCCL-MKL1 cells gave approximately 2 fold more MK colonies than pCCL infected cells at both low cell concentration plating (395 vs. 182 colonies, respectively) and high concentration plating (900 vs. 389 colonies, respectively). These data suggest that overexpression of human MKL1 enhances megakaryocyte differentiation of primary human CD34+ cells. A further understanding of the normal roles of RBM15 and MKL1 in megakaryopoiesis will allow us to better understand the role of the RBM15-MKL1 fusion in AMKL, and aid in the development of treatments for this disease.

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

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

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

Ciclopirox Ethanolamine Is a Novel Modifier of Human Hematopoietic Stem Cell Ex Vivo Expansion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 36-36
Author(s):  
Mehrnaz Safaee Talkhoncheh ◽  
Fredrik Ek ◽  
Aurelie Baudet ◽  
Christine Karlsson ◽  
Roger Olsson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cell Activity ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Cells Cultured

Abstract Despite extensive studies over the last decades, little is known about the mechanisms governing human hematopoietic stem cell (HSC) fate decisions. In particular, it has been challenging to define culture conditions in which HSCs can be expanded for clinical benefit. Application of small molecule screening to modulate stem cells has emerged as a useful tool for identification of new compounds with ability to expand hematopoietic stem and progenitor cells (HSPCs). Such screens have mainly relied on the expression of CD34 as predictor of stem cell activity in cultured cells. However, CD34 defines a broad repertoire of progenitor cells and does not define stem cell function. We found that the long-term repopulation potential of cultured human HSPCs is exclusively contained within a discrete cell population co-expressing CD34 and CD90, while the vast majority of progenitor cells are found in the CD34+CD90- population. Tracking the CD34+ CD90+ population is therefore a sensitive and specific tool to predict stem cell activity in cultured hematopoietic cells and provides a good basis for a screen aimed at discovering modifiers of stem cell expansion. To search broadly for novel and potential modifiers of ex vivo HSCs expansion we next developed and optimized a small molecule screen in human cord blood (CB) derived CD34+ cells. We screened >500 small molecules from 8 different annotated chemical libraries for the phenotypic expansion of CD34+ CD90+ cells following a 6-day culture in serum-free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and fms-like tyrosine kinase 3 ligand (FL). The numbers of CD34+ CD90+ cells for each molecule, tested at two different concentrations, was compared to DMSO treated controls. Following the initial screen, several candidate hits were selected and subjected to a dose response validation experiment from which we selected four top candidate molecules. Two of these molecules were histone deacetylase (HDAC) inhibitors, which recently have been reported to facilitate expansion of CB derived HSCs. One of the top candidates, Ciclopirox ethanolamine (CE), had previously not been implicated in HSC expansion. Ciclopirox ethanolamine is known as an antifungal agent and iron chelator. It has further been shown to suppress cancer cell survival through inhibition of Wnt/beta catenin signaling. We found that CB cells cultured with CE had a 4-fold increase in CD34+90+ cell number compared to DMSO treated controls following 6 days of culture. Interestingly, the total cell count was not different, suggesting a specific increase in CD34+ CD90+ cell number rather than an overall higher proliferation rate. When plated in methylcellulose, CE cultured cells generated increased numbers of myeloid colonies. Moreover, CE treated cells gave rise to multilineage colonies (CFU-GEMM) that could not be detected from the control cultures. To further test the functional capacity of cells cultured with CE, we transplanted cultured equivalents of 30,000 CB CD34+ cells (cultured with or without CE) into sub lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human hematopoietic reconstitution in peripheral blood was determined 16 weeks later. Mice transplanted with CE cultured cells showed higher human CD45 engraftment 16 weeks post transplant compared to control cells (33.2±6.7% vs 14.6±5% p=0.04). The engrafted cells contributed to both myeloid and lymphoid lineages. This shows that Ciclopirox ethanolamine enhances the long-term engraftment capacity of ex vivo cultured HSCs and suggests that it should be considered in stem cell expansion protocols, either alone or in combination with other molecules. We are currently addressing the basis for the increased stem cell activity mediated by Ciclopirox ethanolamine using parameters for differentiation, cell cycling and apoptosis. In addition, we are comparing Ciclopirox ethanolamine with other recently defined modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of Lymphomyeloid Primitive Progenitor Cells in Fresh Human Cord Blood and in the Marrow of Nonobese Diabetic–Severe Combined Immunodeficient (NOD-SCID) Mice Transplanted with Human CD34+ Cord Blood Cells

1999 ◽  
Vol 189 (10) ◽  
pp. 1601-1610 ◽  
Author(s):  
Catherine Robin ◽  
Françoise Pflumio ◽  
William Vainchenker ◽  
Laure Coulombel
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Direct Demonstration ◽  
Cd34 Cells ◽  
Cell Assays ◽  
Cord Blood Cells

Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In this work, we used single cell cultures to show that human cord blood (CB) contains totipotent CD34+ cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34+ CD19−Thy1+ (or CD38−) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on murine stromal feeder layers. 10% of the clones individually analyzed produced CD19+, CD56+, and CD15+ cells in stromal cocultures and CD4+CD8+ T cells in FTOCs, identifying totipotent progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34+ cells. These results provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34+ cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.

scholarly journals Platelet-Derived Stromal Cell–Derived Factor-1 Regulates Adhesion and Promotes Differentiation of Human CD34+Cells to Endothelial Progenitor Cells

Circulation ◽  
2008 ◽  
Vol 117 (2) ◽  
pp. 206-215 ◽  
Author(s):  
Konstantinos Stellos ◽  
Harald Langer ◽  
Karin Daub ◽  
Tanja Schoenberger ◽  
Alexandra Gauss ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Stromal Cell ◽  
Endothelial Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

scholarly journals A Low-Molecular-Weight Antagonist for the Human Thyrotropin Receptor with Therapeutic Potential for Hyperthyroidism

Endocrinology ◽  
2008 ◽  
Vol 149 (12) ◽  
pp. 5945-5950 ◽  
Author(s):  
Susanne Neumann ◽  
Gunnar Kleinau ◽  
Stefano Costanzi ◽  
Susanna Moore ◽  
Jian-kang Jiang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cultured Cells ◽  
Therapeutic Potential ◽  
Partial Agonist ◽  
Primary Cultures ◽  
Graves Disease ◽  
Low Molecular Weight ◽  
Thyrotropin Receptor ◽  
Mrna Transcripts ◽  
Orally Active

Low-molecular-weight (LMW) antagonists for TSH receptor (TSHR) may have therapeutic potential as orally active drugs to block stimulating antibodies (TsAbs) in Graves’ hyperthyroidism. We describe an approach to identify LMW ligands for TSHR based on Org41841, a LMW partial agonist for the LH/choriogonadotropin receptor and TSHR. We used molecular modeling and functional experiments to guide the chemical modification of Org41841. We identified an antagonist (NIDDK/CEB-52) that selectively inhibits activation of TSHR by both TSH and TsAbs. Whereas initially characterized in cultured cells overexpressing TSHRs, the antagonist was also active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs in which it inhibited TSH- and TsAb-induced up-regulation of mRNA transcripts for thyroperoxidase. Our results establish this LMW compound as a lead for the development of higher potency antagonists and serve as proof of principle that LMW ligands that target TSHR could serve as drugs in patients with Graves’ disease.

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