Minimal Engraftment of Human CD34+Cells Mobilized From Healthy Donors in the Infarcted Heart of Athymic Nude Rats

2009 ◽  
Vol 18 (6) ◽  
pp. 845-856 ◽  
Author(s):  
Claus S. Sondergaard ◽  
Jesper Bonde ◽  
Frederik Dagnæs-Hansen ◽  
Jan M. Nielsen ◽  
Vladimir Zachar ◽  
...  
Keyword(s):  
Human Cd34 ◽  
Infarcted Heart ◽  
Nude Rats ◽  
Healthy Donors ◽  
Cd34 Cells

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.

MT1-MMP and RECK Inversely Regulate Hematopoietic Progenitor Cell Egress.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1259-1259
Author(s):  
Abraham Avigdor ◽  
Yaron Vagima ◽  
Polina Goichberg ◽  
Shoham Shivtiel ◽  
Melania Tesio ◽  
...  
Keyword(s):  
Steady State ◽  
Progenitor Cell ◽  
Scid Mice ◽  
Hematopoietic Progenitor Cell ◽  
Chimeric Mice ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.

Preferential Mobilization of CD34+ Plasmacytoid Dendritic Cell Precursors by Plerixafor.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 32-32 ◽  
Author(s):  
Michael P. Rettig ◽  
Kyle McFarland ◽  
Julie Ritchey ◽  
Matthew Holt ◽  
Elena Deych ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Cell Surface ◽  
Cord Blood ◽  
Expression Profiles ◽  
Cell Surface Expression ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
The Impact

Abstract Abstract 32 Background: Plerixafor (Mozobil®) is a CXCR4 antagonist that was recently approved by the Food and Drug Administration for use in combination with granulocyte-colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSCs) in patients with non-Hodgkin's lymphoma and multiple myeloma undergoing autologous transplantation. As part of our ongoing clinical evaluation of plerixafor, we observed that the drug efficiently mobilized a CD34dim population of HSCs. Here, we characterize the CD34dim population and assess its relative frequency following mobilization with G-CSF and/or plerixafor in apheresis samples and in non-mobilized cord blood samples. Methods: Aliquots of apheresis products were obtained from 16 healthy donors following treatment with a single injection of plerixafor or 5 days of G-CSF (10 μg/kg/day). In a separate study, five patients with lymphoma were mobilized sequentially with plerixafor (240 μg/kg), G-CSF (10 μg/kg/day), and plerixafor + G-CSF. These patients received an injection of plerixafor on day −5. Twenty-four hours later, patients were treated with G-CSF for 4 days. On the fifth day (day 0), patients received a dose of G-CSF and a dose of plerixafor followed four hours later by apheresis. Samples were collected after treatment with plerixafor on day −5 as well as immediately before and 4 hrs after plerixafor administration on day 0. Cord blood units were obtained from the St. Louis Cord Blood Bank. Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection CD34 isolation kit. We used Affymetrix Human Genome U133+2 arrays to generate gene expression profiles for 24 samples of CD34+ cells collected following mobilization of healthy donors with either Plerixafor (n=12) or G-CSF (n=12). Results: Human CD34+ cells can be divided into three distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Rα): (i.) CD34+CD45RA−CD123+/− primitive HSCs, (ii.) CD34+CD45RA+CD123+/− committed progenitors, and (iii.) CD34dimCD45RA+CD123hi cells. Table 1 shows the relative frequencies of each of these CD34+ cell subsets in apheresis products obtained from healthy donors mobilized with G-CSF or plerixafor as well as in cord blood units. Strikingly, we observed that each graft type was significantly enriched for one of the CD34+ cell subsets compared to the other two grafts. G-CSF mobilized grafts contained more CD45RA−CD123+/− primitive HSCs, cord blood units were enriched with CD45RA+CD123+/− committed progenitors and plerixafor mobilized products had significantly more CD34dimCD45RA+CD123hi cells. Table 1 also shows the CD34 cell subset distribution following sequential mobilization of lymphoma donors with plerixafor, G-CSF, and plerixafor + G-CSF (PL+G). This data agrees with the results we obtained from healthy donors and confirms that G-CSF grafts are enriched with CD34+CD45RA−CD123+/− cells while plerixafor preferentially mobilizes the CD34dimCD45RA+CD123hi subset. Extensive FACS and functional analyses determined that the CD34dim population represents a plasmacytoid pro-DC2 (for progenitor of pre-dendritic cell type 2) progenitor compartment as indicated by their CD45RA+CD123hiBDCA−2+BDCA−4+CD36+CXCR4hiCD4dimCD25−CD13− phenotype and inability to form CFU-GM colonies in vitro. Finally, we found that the gene signature of plerixafor-mobilized CD34+ cells is distinct from that of G-CSF-mobilized CD34+ cells. Plerixafor-mobilized CD34+ cells expressed significantly more of the specific transcriptional regulator of plasmacytoid DC (pDC) development, E2-2, as well as additional transcriptional factors (SpiB, IRF7, IRF8) and cell surface markers (BDCA-2, ILT7) that are specific to the pDC lineage. Conclusions: This data suggest that the CD34dimCD45RA+CD123hi cells preferentially mobilized by plerixafor are precursor pDCs. Further study is required to determine the impact these cells have on the engraftment and function of plerixafor mobilized grafts after hematopoietic stem cell transplantation. Disclosures: DiPersio: Genzyme Corp.: Honoraria.

Characterization of Human CD34+ Hematopoietic Stem Cells Following Administration of G-CSF or Plerixafor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3476-3476 ◽  
Author(s):  
Michael P. Rettig ◽  
William D. Shannon ◽  
Julie Ritchey ◽  
Matthew Holt ◽  
Kyle McFarland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Hematopoietic Stem Cells ◽  
B Lymphocytes ◽  
Gene Signature ◽  
Drug Clearance ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Background: Current clinical protocols use granulocyte colony-stimulating factor (G-CSF) to mobilize normal hematopoietic stem cells (HSCs) from the bone marrow (BM) to the peripheral blood. Unfortunately, this process requires from 4 to 6 days of G-CSF injection and is associated with significant morbidity, most notably bone pain. We are evaluating a novel method for the mobilization of HSCs using a direct antagonist of the CXCR4/SDF-1 interaction called Plerixafor (AMD3100). Methods: Human CD34+ cells were collected from three different studies at our institution. In the first study, fifteen healthy allogeneic related donors were initially mobilized with increasing doses of intravenous (IV) AMD3100 (80, 160, 240, or 320 μg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single subcutaneous (s.c.) dose of 240 μg/kg AMD3100 and collected cells were used as a source of HSCs for transplantation. In the second study, ten healthy donors were mobilized with 5 days of s.c. injection of G-CSF (10 μg/kg/day), and leukapheresed on day 5. In the final study, eight individual normal donors were mobilized sequentially with AMD3100 and G-CSF. Donors received 1 s.c. injection of 240 μg/kg AMD3100, followed by leukaphersis beginning 4 h after drug treatment. After 10 days of drug clearance, the same donors were mobilized with 5 days s.c. injection of 10 μg/kg/day G-CSF, and leukapheresed on day 5. Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection (MACS) CD34 isolation kit and total RNA was isolated using RNeasy Mini Kit columns (Qiagen). The purity (>85% for all experiments) and phenotype of isolated CD34+ cells was quantified by flow cytometry. RNA profiling analyses were performed using Affymatrix U133+2 arrays. Results: Peak mobilization of CD34+ cells occurred 4 to 6 hours after both IV and s.c. dosing, however, patients given IV doses had higher peak levels of CD34/μl at every time point. There was a clear doseresponse relationship of IV AMD3100 on mobilization of CD34+ HSCs in normal donors, with the 320 μg/kg dose yielding a maximum increase in circulating CD34+ cells from 3.3 ± 1.8 CD34+/μl at baseline to 28.8 ± 4.7 CD34+/μl at 6 h after injection. Although the magnitude of neutrophil, monocyte, and T lymphocyte mobilization by IV AMD3100 was less than that observed for CD34+ cells (2 to 3 fold increase over baseline), the kinetics of their mobilization were similar to the CD34+ HSCs (peak mobilization 4 to 6 h after AMD3100). In contrast, B-lymphocytes were mobilized more rapidly (4.5 ± 1.7-fold at 15 min post-AMD3100) and efficiently (6.6 ± 2.6-fold at 2 h post-AMD3100) by IV AMD3100. This rapid mobilization of B-lymphocytes correlates with our pharmacokinetic studies, which showed that peak levels of AMD3100 occur between 15 and 30 minutes after IV infusion. The gene signature of AMD3100-mobilized human CD34+ HSCs is distinct from that of G-CSF-mobilized CD34+ cells. Of note, EMR1, GIMAP8, PIM1, S100A8, SOCS3, and TMEM49 were expressed more abundantly in all GCSF-mobilized CD34+ cells while BCL-2, CLC, CXCR4, C200rf118, DNTT, IRF8, PRG2, RASD1, RNASE6, and UHRF1 were more abundantly expressed in all AMD3100-mobilized CD34+ cells. Interestingly, the RNA profile of CD34+ HSCs obtained from the BM of three healthy donors clustered with AMD3100-mobilized CD34+ HSCs rather than GCSF mobilized HSCs. Using flow cytometry, we identified a CD34dimCD45RA+ hematopoietic precursor cell that is uniquely enriched in nearly 60% of the AMD3100 products evaluated to date (6/10 patients). In contrast to G-CSF mobilized products, where <2% of CD34+ cells are CD34dimCD45RA+, up to 20% of CD34+ cells in AMD3100 treated donors are CD34dimCD45RA+. Preliminary flow cytometry data suggest that this CD34dimCD45RA+ population represents a pro-DC2 (for progenitor of pre-dendritic cell type 2) progenitor compartment as indicated by their IL-3RαbrightCD62Lbrightα4β7dimCD4dimCD25−c-kit−CD13− phenotype. Conclusions: These observations suggest that IV AMD3100 may be a more effective mobilization agent with a low side effect profile. The gene signature and phenotype of AMD3100-mobilized CD34+ HSCs is distinct from that of G-CSF-mobilized HSCs and resemble CD34+ HSCs present in an unmanipulated BM.

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1585-1593 ◽  
Author(s):  
Anna Jetmore ◽  
P. Artur Plett ◽  
Xia Tong ◽  
Frances M. Wolber ◽  
Robert Breese ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
G1 Cells ◽  
Active Proliferation

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34+ cells, or their G0(G0CD34+) or G1(G1CD34+) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34+ cells harbored similar numbers of CD34+ and CD34− cells, indicating that CD34+ cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G2+M than in recipients of G0/G1 cells (34.6% ± 5.9% and 17.1% ± 6.3%, respectively; P &lt; .01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.

Intrathymic and extrathymic development of human plasmacytoid dendritic cell precursors in vivo

Blood ◽  
2002 ◽  
Vol 99 (8) ◽  
pp. 2752-2759 ◽  
Author(s):  
Kees Weijer ◽  
Christel H. Uittenbogaart ◽  
Arie Voordouw ◽  
Franka Couwenberg ◽  
Jurgen Seppen ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Fetal Liver ◽  
Plasmacytoid Dendritic Cell ◽  
T And B Cells ◽  
Human Thymus ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Fetal Liver Cells ◽  
Thymus Graft

Abstract The development of plasmacytoid dendritic cells (pDC2) from human CD34+ stem cells in vivo was studied in RAG-2−/− interleukin (IL)-2Rγ−/− mice that lack functional T and B cells and natural killer cells. CD34+ cells isolated from fetal liver or thymus were labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and were injected into a human thymus grafted subcutaneously in the RAG-2−/− IL-2Rγ−/− mice. One to 4 weeks later the CFSE label was found not only in T cells but also in CD123+/high CD4+CD45RA+ pDC2, indicating that the CD34+ cells can develop into pDC2 within a thymus. In addition to pDC2, CFSE-labeled dendritic cells with a mature phenotype, determined by the cell surface markers CD11c, CD83, and CD80, were found in the injected human thymus graft. pDC2 was not found in the periphery of mice carrying a human thymic graft, indicating that the intrathymic pDC2 failed to emigrate from the thymus. We also demonstrate that pDC2 can develop outside the thymus because relatively high percentages of pDC2 were found in the periphery after the intravenous injection of CD34+CD38−fetal liver cells in RAG-2−/− IL-2Rγ−/−mice without a human thymus graft. These data indicate that the thymus and the peripheral pDC2 develop independently of each other.

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