Longitudinal Trends in Peripheral Blood Parameters Predict Development of Therapy-Related Myelodysplasia/Acute Myeloid Leukemia (t-MDS/ AML) after Autologous Transplantation for Lymphoma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2133-2133
Author(s):  
Liton Francisco ◽  
Can-Lan Sun ◽  
Lester Laddaran ◽  
Melanie Sabado ◽  
Alysia Bosworth ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Blood Parameters ◽  
Red Cell ◽  
Hematopoietic Stem ◽  
Time Points ◽  
Over Time

Abstract t-MDS/AML is the most common cause of non-relapse mortality in patients undergoing autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL). Although t-MDS/AML is known to result from damage to hematopoietic stem cells (HSC) as a result of genotoxic cancer treatment, the sequential cellular and molecular changes leading to its development are not clearly defined. To better understand the pathogenesis of t-MDS/AML, we conducted a prospective study in 179 patients undergoing aHCT for HL (n=41) or NHL (n=138) between 1999 and 2004, who participated in a prospective longitudinal study from pre-aHCT to five years post-aHCT, with a serial collection of bone marrow and peripheral blood samples. The median length of follow-up for this cohort was 3.9 years. This report focuses on alterations in peripheral blood parameters from pre-aHCT to the development of t-MDS/AML, and compares these trends with the patients in this cohort who did not develop t-MDS/AML. A total of 22 patients have developed t-MDS/AML in this longitudinally followed cohort thus far, resulting in a cumulative incidence of 11% at 5 years. Serial evaluation of peripheral blood parameters including hematocrit, mean corpuscular volume (MCV), hemoglobin (HGB), red cell distribution width (RDW), white blood cell (WBC) count, and platelet (PLT) count, were abstracted from medical records for the following time points: pre-aHCT, day 100, 6 month, 1 year, 2 year, 3 year, 4 year and 5 year after aHCT, for a total of 1129 time points. Values of peripheral blood parameters associated with post-aHCT relapse or persistence of the primary lymphoma or from 3 months prior to development of t-MDS/AML, were excluded from analysis. As shown in the Figure, comparison of the peripheral blood parameters in subjects who developed t-MDS/AML (cases; n=22) with those who did not (controls; n=157) revealed that hematocrit values were lower for cases compared to controls at all post-aHCT time points. HGB values were lower among cases compared to controls at all post-aHCT time points. The RDW values were higher for cases compared to controls at day 100, 6 months and 1 year post-aHCT. MCV values did not differ between cases and controls at any of the time points. WBC counts for the cases were lower than controls pre-aHCT and also at all time points from 6 months post-aHCT onwards. PLT counts for cases were lower than controls at all time points pre- and post-aHCT. A fixed effect growth curve model was fitted to the data from day 100 to 5 years post-aHCT after adjusting for age at aHCT, primary diagnosis, race/ethnicity, and sex, to examine the rate of change in the peripheral blood parameters over time. Results revealed a significantly sharper decline in MCV for cases (β per 100 days = −0.43) over time as compared to controls (β =−0.15; p = 0.006). Although hematocrit increased with time for both cases and controls, the slope for the cases was significantly less steep (controls: β per 100 days=0.31 vs. cases: β per 100 days=0.12; p =0.01). In summary, we consistently observed lower values for red cell parameters, WBC, and platelets in patients with t-MDS/ AML as compared to controls across multiple timepoints post-aHCT. These differences appeared soon after HCT, were persistent, and preceded the development of t-MDS/AML. Our previous studies indicate that there is increased turnover and reduced regenerative capacity of premalignant hematopoietic stem cells at early stages of development of t-MDS/AML. The early and persistent reduction in peripheral blood parameters observed here provides further evidence that bone marrow injury and ineffective hematopoiesis long predate the development of t-MDS/AML after aHCT. Poor hematocrit recovery and enhanced decline in MCV after aHCT were independently associated with increased risk of t-MDS/AML and warrant further development as readily applied biomarkers for disease and the need for close monitoring. Figure Figure

The Value of Bone Marrow Biopsy in the Mobilization of Hematopoietic Stem Cells into the Peripheral Blood in Hematologic Malignancies.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4113-4113
Author(s):  
Leandro De Padua Silva ◽  
Karin Z. Cecyn ◽  
Maria Regina R. Silva ◽  
Jose Salvador R. Oliveira
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Hematologic Malignancies ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Mean

Abstract Background: The mobilization the Peripheral Blood Stem Cells (PBSC) from bone marrow (BM) to peripheral blood (PB) is a complex process yet. This process is related to mobilization growth factor and/or chemotherapy protocol and disease status. CXCR4, receptor of SDF-1 and VLA-4 searched in primitive CD34+ and CD34+Thy+ cells have shown some functions in migration of CD34+ cells. Marrow disease infiltration and grade of fibrosis seem to be important features related to CD34+ cells trafficking from BM to PB. The objective of this study was to compare the cellularities, degrees of disease infiltration, and grades of fibrosis of hematopoietic stem cells (HSCs) in BM biopsies before and after mobilization and to correlate the results with the success of the mobilization in patients with hematologic malignancies and in donors. Patients and Methods: Fifty-nine patients and 21 PB stem cell (PBSC) donors participated in this prospective study between January/2003 and May/2006. The patients included 21 with non-Hodgkin lymphoma (NHL), 21 with multiple myeloma (MM), 10 with Hodgkin lymphoma (HL), and 7 with leukemia (5 AML, 1 ALL, and 1 CLL). The mobilization regimen consisted of 5 g/m2 of cyclophosphamide in 29 patients (21 MM, 4 NHL, 3 HL, and 1 CLL), DHAP in 8 (7 NHL and 1 NHL), ICE in 14 (9 NHL and 5 HL), high-dose Ara-C in 5 (all with AML), and G-CSF in 24 (21 donors, 1 NHL, 1 HL, and 1 ALL). The mean numbesr for previous chemotherapy cycles were 11, 10, 6, and 5 for NHL, HL, MM, and leukemia, respectively. We collected PBSC using a Spectra-Cobe device (Cobe, Lakewood, CO, USA). Large-volume leukoapheresis (LVL) was used in all cases. Mobilization success was defined as > 8 × 106 CD34+ cells/L in PB before collection, and adequate LVL yield was defined as 2 × 106 CD34+ cells/kg in LVL products. Overall BM and WBC precursor cellularities were visually assessed as normal (20–50%), decreased (<20%), and increased (> 50%). Fibrosis was graded as absent, slight (grades 1/2), or severe (grades 3/4). The clonal patterns of infiltration were confirmed by immunohistochemicaly analyses using monoclonal antibodies. Results: Eighty proposals (59 patients and 21 donors) were presented for mobilization. There were 49 men and 31 women; the median age and mean duration of disease at time of mobilization were 40.3 years (range 25–68 years) and 15.9 months (range 10.3–20.1 months). Eighteen patients were complete responders, 37 were partial responders, and 4 had refractory disease. The median duration of G-CSF usage was 5 days. Mobilization failed in 17 patients (2 MM, 8 NHL, 3 HL, and 4 leukemia) and 1 donor. The mean numbers of CD+34 cells/μL in the PB at time of LVL were 110 in MM (1.1–647), 64.5 in NHL (0–211), 29.3 in HL (2–80), and 16.2 in leukemia (1.3–65). The mean yield of PBSC × 106/Kg in LVL were 15.3 in MM (1.1–85.5), 9.4 in NHL (2.4–26.6), 6.3 in HL (1.3–14.1), 6.1 in donors (1.9–11.9), and 5.0 in leukemia (2.9–7.3). There were correlations between successful mobilization and high overall cellularity pre- and post-mobilization (p=0.015 and p=0.002, respectfully) and successful mobilization and high WBC precursors post-mobilization (p=0.009). Conclusion: BM biopsy is a method important to re-statement at pre transplant, should be continue searched by overall and WBS precursor’s celularities, fibrosis and clonal infiltration before mobilization.

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
Author(s):  
Viktor Janzen ◽  
Heather E. Fleming ◽  
Michael T. Waring ◽  
Craig D. Milne ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

The Prognostic Role of Jagged-1 Protein Expression In Acute Myeloid Leukemia Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2726-2726 ◽  
Author(s):  
Agnieszka Wierzbowska ◽  
Agnieszka Pluta ◽  
Konrad Stepka ◽  
Magdalena Czemerska ◽  
Barbara Cebula-Obrzut ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Protein Expression ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Univariate Analysis ◽  
Response To Treatment ◽  
Notch 1 ◽  
Hematopoietic Stem

Abstract Abstract 2726 Objectives: Jagged-1 is a member of the Delta/Serrate/Lag-2 (DSL) family of proteins that are ligands for Notch receptors. Aberrant Jagged-1/Notch-1 signaling is posited to promote the development of AML by inducing excessive self-renewal with a concomitant block in cell differentiation. Moreover, Notch-1 signaling has been identified as a critical factor involved in the maintenance of a pool of self-renewing hematopoietic stem cells (HSC) as well as AML stem cells. So far there were no reports on the clinical role of Notch-1 and Jagged-1 expression in AML. In this study we evaluated the expression of Jagged-1 and Notch-1 proteins in AML blasts and CD34+ peripheral blood stem cells (PBSC) collected during mobilization procedures before autologous stem cell transplantation. In addition, in AML patients we correlated the expression of both proteins with known prognostic factors and response to treatment. Methods: The expression of Notch-1 and Jagged-1 proteins was examined in leukemic blasts isolated from bone marrow or peripheral blood of 53 de novo AML patients with median age 57 years (range 21–82). CD34+ collected from 13 lymphoma patients (11 multiple myeloma, 1 Hodgkin lymphoma, 1 non-Hodgkin lymphoma) with median age 57 (range 21–69 years) served as a control. All measurements were carried out using multi-colour flow cytometry. In parallel, the isotype controls were performed for all measurements. Protein expression was assessed as a percentage of Notch-1 and Jagged-1 positive cells. The cut-off 20% was used to subdivide patients into “low-expressers” and “high-expressers” group. Results: We found that the median expression of Jagged-1 was significantly higher in AML blasts (18,2%; range 0,9–62,4%) as compared to CD34+ PBSC (3,0%; range 0,9–21%); p<0.0001. In contrast, the expression of Notch-1 in AML patients (median 1,4%; range 0,1–24,8%) was lower than in the control CD34+ cells (median 3,85%; range 0,7–16%); p<0.004. There was no correlation between Jagged-1 and Notch-1 protein expression in both AML blasts and PBSC. Jagged-1 expression was significantly higher in AML patients with WBC ≤20G/L (median 21,2%) compared to group with WBC >20 G/L (median 9,85%); p<0.004. Consequently, we found the significant negative correlation between Jagged-1 expression and WBC count (p<0.02). Patients with good-risk karyotype according to SWOG classification showed significantly higher expression of Jagged-1 protein as compared to intermediate and poor risk group (medians 21,8% vs. 11,5% respectively; p< 0.02). Thirty two out of 53 AML patients received standard induction chemotherapy with daunorubicine and cytarabine (“3+7”), 21/53 received non-intensive therapy. Nineteen (61%) of intensively treated patients achieved complete remission (CR). We observed that the CR rate in “high-expressers” of Jagged-1 was significantly higher than in the “low-expressers” group (80% vs. 43% respectively; p=0.04). Additionally, a good karyotype and a high expression of Jagged-1 protein were the only factors associated with higher probability of CR (p=0.05, p<0.01, respectively) in univariate analysis. There was no statistical association between the Notch-1 expression and response to treatment, karyotype or tumour size associated risk factors as: WBC, percentage of leukemic blasts in bone marrow and LDH. Moreover, no correlations between Notch-1 and CD34 expression as well as Notch-1 and differentiation markers (CD13, CD14, CD15, CD33) expressions in AML blasts were found. Conclusions: Jagged-1 protein is highly expressed in AML blasts and correlates with better response to standard chemotherapy, favorable karyotype and lower WBC in AML patients. These data clearly demonstrate an important role of Jagged-1 in AML biology. A better understanding of autonomous Jadded-1 signaling in AML may create new options for therapeutic interventions in AML. Disclosures: Robak: Johnson & Johnson: Research Funding.

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.

Marking of Embryonic Precursors of Adult Hematopoietic Stem Cells At Gestational Day E7.5-E8.5 Using An Abcg2-CreERT2 Lineage Tracing Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1287-1287
Author(s):  
Sheng Zhou ◽  
Soghra Fatima ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Lineage Tracing ◽  
Tamoxifen Treatment ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Tissue Specific ◽  
Tissue Specific Expression

Abstract Abstract 1287 Abcg2 is a member of the ATP-binding cassette transporter family, is expressed in adult hematopoietic stem cells (HSCs), and is required for the side population phenotype of adult bone marrow HSCs as well as other adult tissue-specific stem cells. We have used these properties of Abcg2 expression for lineage tracing of stem cell development in mice, particularly for HSCs. An ires-CreERT2 cassette was inserted into the Abcg2 locus, down-stream of its stop codon but upstream of its endogenous polyadenylation site, so that both Abcg2 and CreER are co-expressed from a single bicistronic transcript. This design allows for minimum disruption of Abcg2 expression and tissue specific expression of CreERT2 under the control of endogenous Abcg2 transcription elements. The Abcg2CreER/CreER mouse was crossed with flox-STOP-flox-YFP (Rosa26RYFP/YFP) mouse to generate compound heterozygous Abcg2CreER/+ Rosa26RYFP/+ mice. Treatment of adult Abcg2CreER/+ Rosa26RYFP/+ mice with tamoxifen resulted in robust YFP expression in kidney proximal tubule cells and hepatocytes demonstrating the expected tissue-specific expression of the Abcg2CreER allele. We also observed tamoxifen-dependent appearance of YFP+ cells in all hematopoietic lineages in the peripheral blood and bone marrow, confirming our prior observations that Abcg2 is expressed in adult stem cells. Unexpectedly, we observed long term marking in intestinal epithelial cells and in seminiferous tubules 9 to 20 months after tamoxifen treatment, recapitulating classic progeny tracking patterns, proving that intestinal stem cells and spermatogonial stem cells express the Abcg2 marker. Pregnant females were treated with a single dose of 4-hydroxytamoxifen (4-HT) at gestational days E7.5 and E8.5 using overnight timed breeding pairs. We chose 4-HT rather than tamoxifen because 4-HT has been shown to decay relatively quickly in the fetus so that no recombination can occur 24 hours after the pulse. After maternal treatment, mice were born, grew to adulthood, and were analyzed 14–17 weeks after birth for expression of YFP in distinct peripheral blood lineages. In the majority of the 18 mice born from mothers treated with 4-HT at day E7.5 and from 17 mice born of mothers treated at day E8.5, a small but distinct YFP+ subpopulation could be clearly detected in all hematopoietic lineages (Figure A and B). The numbers of marked cells have been stable for approximately 4 months and are strictly dependent on 4-HT treatment of the mother. These results demonstrate that a precursor to adult hematopoietic stem cells exists at gestational day E7.5 to E8.5 and contributes to a stable subpopulation of HSCs well into adulthood. The low level of marking could reflect inefficient recombination due to either relatively low levels of expression of the recombinant allele in these embryonic HSC precursors or due to inefficient nuclear localization with the single 4-HT pulse. Alternatively, these marked embryonic HSC precursors may be generating only a minor population of adult HSCs that are competing against a larger fraction of HSCs that arise from precursors that originate later in gestation after the 24 hour 4-HT washout. We are in the process of determining the embryonic source of the E7.5 – E8.5 adult HSC precursor and have not yet determined whether it originates in the yolk sac, in another extra-embryonic source, or within the embryo proper. We are following these mice for longer periods of time to determine the stability of marking in primary and serial transplant experiments. Altogether, we expect that studies with this novel lineage tracing model will provide a better understanding of steady-state, uninterrupted embryonic hematopoietic development that does not require transplant assays to detect HSC activity. Disclosures: No relevant conflicts of interest to declare.

Levels of the c-Myb Protein Indicate Repopulating Capacity in Long-Term Hematopoietic Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1196-1196
Author(s):  
Hiroshi Sakamoto ◽  
Naoki Takeda ◽  
Kiyomi Tsuji-Tamura ◽  
Saeka Hirota ◽  
Ogawa Minetaro
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Conditional Knockout ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Myb Protein ◽  
Over Time

Abstract Abstract 1196 c-Myb is a transcription factor essential for the proliferation of hematopoietic cells: conventional c-myb deficient mice died around E14 when their hematopoietic progenitors/stem cells fail to proliferate in the fetal livers. Recently, c-myb has also been reported to be crucial for the differentiation of hematopoietic progenitors. We have previously reported that the differentiation into erythrocytes, megakaryocytes and B-lymphocytes is regulated by c-myb levels utilizing ES cell in vitro differentiation combined with a tetracycline-inducible gene expression system. The gene-altered c-myb mice, such as knockdown or conditional knockout mice in the hematopoietic cell lineages, showed that c-myb controlled hematopoietic stem cells (HSCs). In order to examine the levels of the c-Myb protein in HSCs, we established c-Myb reporter mice in which the EGFP cDNA was linked to the coding sequence of the c-myb gene (c-MybEGFP). Homozygous c-MybEGFP mice, showing normal hematopoiesis, expressed EGFP in hematopoietic progenitors. EGFP+ cells were observed in most long-term (LT) HSCs (90–95%), which were defined as CD34− Lin− Sca-1+c-Kithigh cells (34LSKs), CD150+CD48−LSKs and side-population LSKs. To evaluate c-Myb function in LT-HSCs, we transplanted 100 cells of EGFPlow and EGFPhigh of 34LSKs into irradiated mice along with competitor cells (0×106 cells). Both LT-HSC populations presented multilineage repopulating capacity over 20 weeks. In addition, the EGFPlow cells indicated higher chimerism in the total peripheral blood than the EGFPhigh cells at any given time point. The contribution of the EGFPlow-derived cells in the peripheral blood of the recipient mice increased over time whereas EGFPhigh progeny gradually decreased over time. Under a stringent transplantation condition (30 donor cells with 0.4×106 competitor cells), 83.3% of the recipients that received the EGFPlow34LSK showed donor-derived progeny while the EGFPhigh were lower (20.0%) 8 weeks after transplantation. At Week 12, all the recipients with the EGFPlow34LSKs demonstrated donor-derived progeny; however, EGFPhigh 34LSKs-derived cells disappeared totally in all the transplants. These results suggest that the EGFPlow and the EGFPhigh cells in LT-HSCs possess distinct repopulating capacity: the EGFPlow cells are high and the EGFPhigh cells are low. To investigate the relationship between the EGFPlow and the EGFPhigh LT-HSC, we examined EGFP expression levels in the recipient mice grafted EGFPlow34KSL at least 24 weeks after transplantation. EGFPlow34LSK generated EGFPhigh cells in the donor-derived 34LSK population in the recipient mice, suggesting the possibility that the EGFPlow LT-HSCs support the production of the EGFPhigh LT-HSCs. In conclusion, we found that the expression levels of c-Myb protein subdivide LT-HSC population in correspondence with their respective multilineage repopulating capacities. Disclosures: No relevant conflicts of interest to declare.

In Utero Depletion Of Fetal Host Hematopoietic Stem Cells Improves Engraftment Following Neonatal Transplantation In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3240-3240
Author(s):  
Chris Derderian ◽  
Charmin King ◽  
Priya Togarrati ◽  
Agnieszka Czechowicz ◽  
Ninnia Lescano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Hematopoietic Cell Transplantation ◽  
Mononuclear Cells ◽  
Fetal Liver ◽  
In Utero ◽  
Hematopoietic Stem ◽  
In Utero Transplantation

Abstract Introduction In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to treat congenital disorders as the fetal host can potentially be tolerized to transplanted cells early in gestation. However, levels of engraftment have been low and fetal host conditioning strategies to increase space in hematopoietic niches have not been widely explored. We hypothesized that depletion of fetal host hematopoietic stem cells (HSC) using an antibody against the c-kit receptor (ACK2), a strategy which selectively depletes HSC by disrupting stem cell factor (SCF) signaling, would improve engraftment after HSC transplantation. Methods Fetal C57B6.CD45.2 (B6) mice were injected with increasing doses of ACK2 (2.5-50 µg/fetus) or isotype control antibody on E14.5 and surviving pups were transplanted with congenic B6.CD45.1 fetal liver mononuclear cells (2.5×106 cells/pup) on day of life 1 (P1, 7 days after in utero injection), allowing post-transplantation host monitoring. Host HSC depletion and residual serum ACK2 concentration were examined on P1. Peripheral blood chimerism, defined as donor/(donor+host) CD45 cells, as well as the lineage distribution of chimeric cells, were determined beginning 4 weeks after transplantation. Results Survival to birth among fetuses injected with 2.5, 5, or 10 µg of ACK2 was similar to controls (control: 74%; 2.5 µg: 80%; 5 µg: 71%; 10 µg: 60%, p=0.2 by chi-square test, n≥45/group) but was significantly lower at higher concentrations (20 µg: 37%; 50 µg: 31%, p<0.001 vs. control, n≥70/group). Transient anemia and leukopenia were observed on P1 with doses ≥ 5 µg which resolved by P7 (n=17). Four of 19 pups previously treated with ACK2 (2.5-10 µg) and observed long-term had patchy coat discoloration, possibly a manifestation of disruption of C-kit+ melanocyte migration. In utero ACK2 treatment resulted in significant and dose-dependent depletion of host HSCs (defined as Lin-Sca-1+C-kit+, KLS) in the bone marrow of treated animals by P1 (Figure 1A). There was no depletion of KLS cells in the liver. Residual ACK2 antibody was undetectable in the serum by P1, validating our strategy of in utero depletion and neonatal transplantation. In animals receiving neonatal transplantation, ACK2 depletion resulted in a significant increase in levels of engraftment 4 weeks after transplantation compared to controls (control: 3.3±0.3%; 2.5 µg: 13±1.4%; 5 µg: 10±2.4%; 10 µg: 11±2.0%, p<0.05 for each dose vs control by ANOVA). Accordingly, we detected an increased number total bone marrow KLS cells 7 days after transplantation in ACK2 treated animals compared to controls (412±45.9 vs. 933±112 cells, p=0.01, n≥3/group). Moreover, levels of chimerism increased over time in treated animals (Figure 1B; 12 weeks: 2.5 µg: 190%; 5 µg: 170%; 10 µg: 160%) while they remained unchanged in controls. Overall, levels of chimerism achieved with ACK2 treatment were significantly higher than that observed in animals that received in utero transplantation without ACK2 depletion. Lineage analysis of peripheral blood for granulocytes, B cells, and T cells indicated an equal increase in all lineages, suggesting ACK2 depletes true HSCs and not committed progenitors. Interestingly, ACK2 depletion at doses 2.5-10 µg did not result in engraftment of allogeneic BALB/c cells (n=11), indicating that allogeneic neonatal transplantation, unlike in utero transplantation, is limited by a host immune response which is unaffected by ACK2. Conclusion We have demonstrated that fetal HSC depletion using ACK2 can lead to clinically relevant levels of donor cell engraftment with minimal toxicity. In previous studies with this antibody, host HSC depletion required either immunodeficient animals or concurrent irradiation, whereas we achieved depletion in wild-type fetal hosts, suggesting differences in fetal vs. adult HSC sensitivity to SCF signaling. Future studies should explore this strategy to improve engraftment in large animals models of IUHCTx. Disclosures: Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Impact of Infusion Rate on the Early Engraftment Following Allogeneic Intra Bone Marrow Infusion of Hematopoietic Stem Cells in a Canine DLA-Identical Reduced Intensity Transplantation Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5406-5406
Author(s):  
Stephanie Schaefer ◽  
Juliane Werner ◽  
Sandra Lange ◽  
Katja Neumann ◽  
Christoph Machka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Infusion Rate ◽  
Conflicts Of Interest ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Time Points ◽  
The Impact ◽  
Reduced Intensity

Abstract Introduction: Direct intra bonemarrow (IBM) infusion of hematopoietic stem cells (HSC) is assumed to improve the homing efficiency and to accelerate the early engraftment in comparison to the conventional intravenous application of HSC. Especially for transplantation of low cell numbers i.e. "weak grafts" that is generally associated with delayed engraftment. The direct infusion of HSC in close proximity to the HSC niche by intra bone marrow transplantation (IBMT) might be a promising way. Whether the HSC infusion rate might influence the homing process and therefore the outcome after IBMT is so far unknown. Aims: Herein, we analyzed in a canine DLA-identical littermate model the impact of different graft infusion rates on the hematopoietic recovery as well as on the engraftment kinetics after IBMT following reduced intensity conditioning. Methods: Recipient dogs received IBMT following a 4.5 Gy total body irradiation (TBI). From day (d) -1 until d+35 Cyclosporin A (15mg/kg) was administered orally twice a day as immunosuppression. For IBM transfusion the graft volume was reduced by buffy coat centrifugation and dogs obtained 2x25 ml simultaneously into the humerus and femur. The infusion rate of the graft was 25ml/10 min in group 1 (IBM10, n = 8) and 25 ml/60 min in group 2 (IBM60, n = 7). A 28 day follow-up is currently available for twelve dogs (IBM10 n = 7; IBM60 n = 5). The development of the peripheral blood mononuclear cell (PBMC) and granulocyte chimerism was tested weekly. Blood count, kidney and liver enzymes were monitored routinely. Results: All animals engrafted. One dog of the IBM10 group died at d+15 (infection) and was therefore not included into analysis. The median number of infused total nucleated cells were in IBM10 4.1*108/kg (range 2.3-6.0*108/kg) and in IBM60 3.2*108/kg (range 1.8-4.4*108/kg; p=0.4). The infused CD34+ numbers were median 3.2*106/kg (range: 1.2-10.0*106/kg; IBM10) and 3.6*106/kg (range: 1.5-6.8*106/kg; IBM60; p=0.7). Time of leukocyte recovery was median d+11 after IBMT in both groups (range: d+4 to d+11, IBM10; d+8 to d+14, IBM60; p= 0.5). Median leukocytes nadirs amounted to 0.2*109/l for IBM10 and 0.3*109/l for IBM60 (p= 0.08). The median duration of leukopenia (<1*109/l) were similar (6d, range: 4-11d, IBM10; 3-9d, IBM60) (p= 0.6). Median platelet nadir was 0*109/l for both cohorts (range: 0.0-7.0*109/l, IBM10; 0.0-1.0*109/l, IBM60). The period of thrombocytopenia (≤20.0*109/l) was significantly prolonged in the IBM60 group (median 10d, range) compared to 5d (range: 3-12d) in the IBM10 group (p=0.05). Donor PBMC chimerisms at d+7, d+14 and d+28 were median 22% (range: 8-34%), 50% (range: 29-53%) and 67% (range: 47-73%) in IBM10. The results of PBMC chimerism for IBM60 were 11% (range: 5-34%), 42% (range: 20-42%) and 59% (range: 44-66%) at these time points (p = n.s.). Donor granulocyte chimerisms of median 33% (range: 11-83%), 100% (range: 58-100%) and 100% (range: 82-100%) were detected at d+7, d+14 and d+28 after HSCT in IBM10, respectively. The granulocyte chimerism in IBM60 amounted to 34% (range: 3-87%), 96% (range: 94-100%) and 98% (range: 96-100%) at the above mentioned time points p=n.s. for all time points). Conclusion: Our data suggest that early granulocyte and PBMC engraftment is not influenced by modification of the HSC infusion rate. However, the period of thrombocytopenia seems to be prolonged following a 60 minutes application. Therefore, longer infusion times in an IBMT setting seem not to be beneficial following toxicity reduced conditioning regimen. Disclosures No relevant conflicts of interest to declare.

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