Human CD34+Cells Mobilized by AMD3100 Demonstrate Enhanced NOD/SCID Repopulating Function Compared to CD34+ Cells Mobilized by Granulocyte Colony Stimulating Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1962-1962 ◽  
Author(s):  
David A. Hess ◽  
Louisa Wirthlin ◽  
Timothy P. Craft ◽  
Jesper Bonde ◽  
Ryan W. Lahey ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Paired Comparison ◽  
Fold Increase ◽  
Human Cells ◽  
Lymphoid Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Interactions between stromal derived factor-1 (SDF-1 or CXCL12), and its receptor CXCR4 regulate hematopoietic stem and progenitor cell retention in the bone marrow. AMD3100, a bicyclam molecule that selectively blocks the interaction between CXCL12 and CXCR4, has recently been used in clinical trials to rapidly mobilize hematopoietic progenitor cells. However, the functional properties of human stem and progenitor cells mobilized with this agent are not well characterized. Here, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized after 5 days of G-CSF treatment. A total of 7 HLA-matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100. After 1 week of drug clearance, the same donor was mobilized with G-CSF, allowing a paired comparison of the repopulating function of cells mobilized by the two agents. Total CD34+ cells mobilized by AMD3100 treatment averaged 1.2±0.4x106 CD34+ cells/kg (range 0.4–2.1x106 CD34+ cells/kg), as compared to G-CSF treatment at 3.2±0.9x106 CD34+ cells/kg (range 1.7–5.7 x106 CD34+ cells/kg). Leukapheresis total mononuclear cell (MNC) fraction or purified CD34+ cells (>90% purity), were isolated and transplanted into sublethally irradiated NOD/SCID mice at varying doses. BM, spleen, and peripheral blood of mice were harvested 7–8 weeks post-transplantation and analyzed by flow cytometry for the presence or absence of engrafting human cells. Low frequency human engraftment events (<0.2% human cells) were confirmed by PCR for P17H8 alpha-satellite human DNA sequences. Injection of 1–40x106 MNC or 0.5–5x105 CD34+ cells produced consistent human engraftment and allowed limiting dilution analysis using Poisson statistics to be performed on paired samples of AMD3100 and G-CSF leukapheresis products from 3 individual patients. The calculated frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 11.5x106 AMD3100-mobilized MNC (n=50) compared to 1 SRC in 44.8x106 G-CSF-mobilized MNC (n=55). For purified CD34+ populations, the overall frequency of repopulating cells was 1 SRC in 1.0x105 AMD3100-mobilized CDC34+ cells (n=53) compared to 1 SRC in 3.1x105 G-CSF-mobilized CD34+ cells (n=45). These data correspond to a 3–4-fold increase in overall repopulating function demonstrated by AMD3100 mobilized cells. Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with equivalent production of myelo-monocytic cells (CD33+CD14+), immature B-lymphoid cells (CD19+CD20+), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. These studies indicate that human AMD3100-mobilized MNC and purified CD34+ cells possess enhanced repopulating capacity, as compared to G-CSF mobilized counterparts from the same donor. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained and highly functional source of repopulating hematopoietic stem cells for clinical transplantation procedures.

Human CD34+ Cells Rapidly Mobilized by AMD3100 Repopulate NOD/SCID Mice with Equivalent Efficiency as CD34+ Cells Mobilized by G-CSF.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1186-1186
Author(s):  
David A. Hess ◽  
Jesper Bonde ◽  
Timothy P. Craft ◽  
Louisa Wirthlin ◽  
John F. DiPersio ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Paired Comparison ◽  
Drug Clearance ◽  
Scid Mice ◽  
Lymphoid Cells ◽  
Additional Patient ◽  
Cxcr4 Antagonist ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Interactions between the chemokine receptor CXCR4 and its ligand, stromal derived factor-1, regulate hematopoietic stem cell migration. The CXCR4 antagonist, AMD3100, has recently been shown to rapidly mobilize primitive hematopoietic cells. However, the functional properties of stem and progenitor cells mobilized with this agent are not well characterized. Thus, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized by 5 days of G-CSF treatment. Healthy, matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100, and produced an enrichment of circulating CD34+ cells to 0.6% of the total mononuclear cells (MNC). After 2 weeks of drug clearance, the same donor was mobilized with G-CSF (0.4% CD34+ cells), allowing a paired comparison of the repopulating function of cells mobilized by these two regimens. Total MNC, CD34+ cells (&gt;95% purity), and lineage depleted (Lin−) cells (48–55% CD34+) were isolated and transplanted into NOD/SCID mice at various doses. Injection of 106–107 (approximately 5x103–5x104 CD34+ cells) AMD3100-mobilized MNC resulted in bone marrow (BM) engraftment in 6 of 10 mice, whereas equal doses of G-CSF mobilized MNC engrafted 3 of 10 mice. Higher cell doses (2x107 MNC, approximately 1x105 CD34+ cells) consistently produced engraftment in the BM, spleen, and peripheral blood of all mice, with higher levels of engraftment with AMD3100-mobilized cells (1.8±0.5%) compared to G-CSF-mobilized cells (0.4±0.05%, p&lt;0.05). Similar analyses performed using purified CD34+ cells revealed similar engraftment frequencies for both leukapheresis products. Transplantation of 5x104–105 AMD3100-mobilized CD34+ cells engrafted 4 of 7 mice and G-CSF-mobilized CD34+ cells engrafted 5 of 10 mice. However, transplantation of 5x105 AMD3100-mobilized CD34+ cells consistently resulted in higher engraftment levels compared to G-CSF-mobilized CD34+ cells (3.4±1.1% versus 0.8±0.4% human cells, p&lt;0.05). Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with production of myeloid cells (CD33+, CD14−CD13+CD66abce+/−), monocytes (CD14+), immature B-lymphoid cells (CD19+/−CD20+/−), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. Similarly, AMD3100 and G-CSF-mobilized Lin− cells produced consistent engraftment in all transplanted mice (n=20), and demonstrated equivalent engraftment levels and multilineage differentiation when directly comparing AMD3100 versus G-CSF-mobilization. Ongoing analysis of additional patient samples will allow direct comparison of these repopulating cells by limiting dilution analysis using Poisson statistics. These preliminary studies indicate that human AMD3100-mobilized CD34+ cells possess at least equivalent repopulating capacity compared to G-CSF mobilized cells, and therefore represent a more rapidly obtainable source of hematopoietic stem cells for clinical transplantation.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

scholarly journals Granzyme B and perforin lytic proteins are expressed in CD34+ peripheral blood progenitor cells mobilized by chemotherapy and granulocyte colony-stimulating factor

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3500-3506 ◽  
Author(s):  
C Berthou ◽  
JP Marolleau ◽  
C Lafaurie ◽  
A Soulie ◽  
L Dal Cortivo ◽  
...  
Keyword(s):  
Cell Line ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Granzyme B ◽  
Cellular Adhesion ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stimulating Factor

Granzyme B and perforin are cytoplasmic granule-associated proteins used by cytotoxic T lymphocytes and natural killer (NK) cells to kill their targets. However, granzyme B gene expression has also been detected in a non-cytotoxic hematopoietic murine multipotent stem cell line, FDCP-Mix. The objective of the present study was to investigate whether granzyme B and perforin could be expressed in human hematopoietic CD34+ cells and if present, discover what their physiologic relevance could be. The primitive CD34+ human cell line KG1a was investigated first and was found to express granzyme B and perforin. Highly purified hematopoietic stem/progenitor cells were then selected using the CD34 surface antigen as marker. Steady-state bone marrow (BM) CD34+ cells did not contain these proteins. Peripheral blood (PB) CD34+ cells, which had been induced to circulate, were also analyzed. After chemotherapy (CT) and granulocyte colony-stimulating factor (G-CSF) treatment, CD34+ cells strongly expressed mRNAs and proteins of granzyme B and perforin. In contrast, CD34+ cells mobilized by G-CSF alone were negative. Western blot analysis further showed that granzyme B and perforin proteins were identical in CD34+ cells and activated PBLs. Such proteins might be implicated in the highly efficient migration of CD34+ stem/progenitor cells from BM to PB after CT and G-CSF treatment. The cellular adhesion mechanisms involved in the BM homing of CD34+ cells are disrupted at least temporarily after CT. The Asp-ase proteolytic activity of granzyme B on extracellular matrix proteins could be used by progenitor cells for their rapid detachment from BM stromal cells and perforin might facilitate their migration across the endothelial cell barrier.

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4040-4040
Author(s):  
Szabolcs Fatrai ◽  
Simon M.G.J. Daenen ◽  
Edo Vellenga ◽  
Jan J. Schuringa
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Fold Increase ◽  
Marrow Stromal Cells ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Disruption of CXCR4 Utilizing AMD3100 Bypasses Mobilization Deficiency Exhibited by CD26−/− Mice and Results in Efficient Mobilization of Myeloid Progenitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3492-3492
Author(s):  
Laura A. Paganessi ◽  
Andrew L. Walker ◽  
Stephanie A. Gregory ◽  
Henry C. Fung ◽  
Kent W. Christopherson
Keyword(s):  
Bone Marrow ◽  
Blood Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Genetically Engineered Mice ◽  
Colony Forming Units ◽  
Myeloid Progenitors

Abstract The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood 2003 and Christopherson, et al Exp Hematol 2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/− mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2 inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2 and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/− mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/− mice as compared to untreated C57BL/6 and CD26−/− mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/− mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1169-1169
Author(s):  
Kam Tong Leung ◽  
Karen Li ◽  
Yorky Tsin Sik Wong ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Scid Mouse ◽  
Conflicts Of Interest ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Disruption of the Normal Hematopoietic Hierarchy Leading to Stem Cell Exhaustion in an Animal Model of Myelodysplastic Syndrome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1698-1698
Author(s):  
Yang Jo Chung ◽  
Peter D. Aplan
Keyword(s):  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Brdu Incorporation ◽  
Compensatory Mechanism ◽  
Self Renewal ◽  
Hematopoietic Stem

The ineffective hematopoiesis that is characteristic of myelodysplastic syndrome (MDS) suggests functional defects of hematopoietic stem and progenitor cells (HSPC). NUP98-HOXD13 (NHD13) transgenic mice recapitulate many features of human MDS such as ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and transformation to acute myeloid leukemia (AML), and have been used as a pre-clinical model for human MDS. NHD13 mice universally develop signs of MDS (e.g., peripheral blood cytopenia, macrocytosis, dysplasia) at approximately 5 months of age, with median survival of 10 months. Two month old NHD13 mice do not show clear evidence of MDS such as peripheral blood cytopenia, dysplasia, or transformation to AML. Bone marrow nucleated cells (BMNC) from two month old NHD13 mice have a modest 1.3-fold increase of lineage negative (LN) BMNCs compared to age matched WT mice. The increased number of LN BMNCs appeared to be primarily due to a 3.4-fold increase of the LN Sca-1+cKit-(LS+Kˉ) cells, an early lymphoid-committed precursor. Lineage negative Sca-1+ c-Kit+ (LSK) cells, which include the most immature, undifferentiated cells, can be divided into five sub populations, based on expression of Flk2, CD150, and CD48. These populations have been designated Long-Term Hematopoietic Stem Cell (LT-HSC), Short-Term HSC, (ST-HSC), and Multi-Potent Progenitor 2, 3, and 4 (MPP2, MPP3, and MPP4) based on functional assays. Two-month old NHD13 mice had decreased MPP4 (5-fold), decreased LT-HSC (3.6-fold) and increased ST-HSC (2.3-fold) compared with the age matched WT mice. The expansion of ST-HSC two-month old NHD13 mice was associated with increased cell proliferation of ST- HSC, as assessed by bromo-deoxy-uridine (BRDU) incorporation. We next studied LSK subsets from NHD13 mice aged seven months, which coincided with peripheral blood findings consistent with MDS (e.g. anemia, thrombocytopenia, macrocytosis), BM from seven month old NHD13 mice showed significant reductions of all LSK population subsets. LT-HSCs show differential expression of the CD41 antigen, and CD41ˉ LT-HSCs are more quiescent than CD41+ LT-HSCs and are thought to reside at the apex of the hematopoietic differentiation hierarchy. Although there was no difference in the absolute number of quiescent CD41ˉ LT-HSC between two and six month old WT mice, six month old NHD13 mice show a marked decrease (4.2 fold) in CD41ˉ LT-HSCs, suggesting exhaustion of LT-HSC in NHD13 mice. Colony forming assays were used to assess function of the five LSK sub-populations in vitro. LT-HSC and ST- HSC from NHD13 BMNC did not produce any colonies in two independent experiments, whereas MPP2 and MPP3 from NHD13 BMNC produced a similar number and lineage distribution of colonies compared to WT BMNC. This result suggested that HSCs from NHD13 BMNC may be functionally impaired, and that NHD13 hematopoietic progenitor cells may instead be derived primarily from MPP2 and MPP3. To evaluate HSC self-renewal activity, the five LSK subsets from NHD13 BMNC were transplanted to lethally irradiated mice together with 5 x 105 WT BMNC competitor cells. None of the NHD13 LSK sub-populations showed evidence of engraftment. Since NHD13 LN BMNC have previously been shown to be more prone to apoptosis than their WT counterpart, it is possible that lack of engraftment of NHD13 LSK subsets was due to the ex vivo sorting procedure. However, we also considered the possibility that NHD13 lineage positive (LP) BMNC had acquired self-renewal potential, and were contributing to long term hematopoiesis in the NHD13 BM. Therefore, we transplanted LP and LN BMNC from NHD13 or WT mice into WT recipients, again with WT competitor BMNC. Almost half of the NHD13 LP recipients showed long-term (>26 weeks) myeloid engraftment, whereas none of the WT LP recipients showed long term myeloid engraftment. Taken together, these findings suggest that the primitive LT-HSC (LSK Flk2ˉ CD150+CD48ˉ CD41ˉ) from NHD13 BM become exhausted with age, corresponding to the presentation of findings consistent with MDS (peripheral blood cytopenia, macrocytosis). Furthermore, self-renewal activity of NHD13 LP BMNCs suggest the existence of a compensatory mechanism for the homeostasis of hematopoiesis in MDS. Disclosures Aplan: NIH: Patents & Royalties: royalties for the invention of NUP98-HOXD13.

scholarly journals Nonobese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) Mouse as a Model System to Study the Engraftment and Mobilization of Human Peripheral Blood Stem Cells

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2556-2570 ◽  
Author(s):  
Johannes C.M. van der Loo ◽  
Helmut Hanenberg ◽  
Ryan J. Cooper ◽  
F.-Y. Luo ◽  
Emmanuel N. Lazaridis ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Severe Combined Immunodeficiency ◽  
Human Cells ◽  
Scid Mice ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Nonobese Diabetic

Mobilized CD34+ cells from human peripheral blood (PB) are increasingly used for hematopoietic stem-cell transplantation. However, the mechanisms involved in the mobilization of human hematopoietic stem and progenitor cells are largely unknown. To study the mobilization of human progenitor cells in an experimental animal model in response to different treatment regimens, we injected intravenously a total of 92 immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with various numbers of granulocyte colony-stimulating factor (G-CSF) –mobilized CD34+ PB cells (ranging from 2 to 50 × 106cells per animal). Engraftment of human cells was detectable for up to 6.5 months after transplantation and, depending on the number of cells injected, reached as high as 96% in the bone marrow (BM), displaying an organ-specific maturation pattern of T- and B-lymphoid and myeloid cells. Among the different mobilization regimens tested, human clonogenic cells could be mobilized from the BM into the PB (P= .019) with a high or low dose of human G-CSF, alone or in combination with human stem-cell factor (SCF), with an average increase of 4.6-fold over control. Therefore, xenotransplantation of human cells in NOD/SCID mice will provide a basis to further study the mechanisms of mobilization and the biology of the mobilized primitive human hematopoietic cell.

scholarly journals Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4332-4332
Author(s):  
Xinxin Huang ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Lentiviral Vectors ◽  
Ex Vivo Expansion ◽  
Transcriptional Factor ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1405-1412 ◽  
Author(s):  
DN Haylock ◽  
LB To ◽  
TL Dowse ◽  
CA Juttner ◽  
PJ Simmons
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells

Abstract Hematopoietic reconstitution (HR) after peripheral blood stem cell transplantation is characterized by a delay of 8 and 12 days for recovery to safe levels of neutrophils and platelets even in patients with the most rapid engraftment. We postulate that a further enhancement in the rate of HR may be achieved by transplanting with an expanded postprogenitor cell population that can provide mature functional cells within days of infusion. In this study we investigated the ability of combinations of hematopoietic growth factors (HGF) to generate nascent granulocyte-macrophage colony-forming units (CFU-GM) in a 7-day suspension culture of peripheral blood CD34+ cells. A combination of 6 HGF, ie, interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage- CSF (GM-CSF), and stem cell factor (SCF), was identified as the most potent combination of those tested. Subsequently, large volume suspension cultures of CD34+ cells from the same patients using the same 6-factor combination were established and monitored for 21 days. An exponential rate of nucleated cell production (mean 1,324-fold increase) occurred during culture. CFU-GM production paralleled nucleated cell production until day 10, peaked at day 14 (mean 66-fold increase), and was then maintained until day 21. Cells produced in culture were predominantly neutrophil precursors and developed normally as assessed by morphology, immunophenotype, and superoxide generation. This stroma-free, cytokine-driven culture system can achieve a degree of amplification, which suggests the feasibility of ex vivo culture of hematopoietic progenitor cells as an adjunct to hematopoietic stem cell transplantation.

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