Alginate-Chitosan Microencapsulated Cells for Improving CD34+ Progenitor Maintenance and Expansion

Retno Wahyu Nurhayati; Rafianto Dwi Cahyo; Gita Pratama; Dian Anggraini; Wildan Mubarok; Mime Kobayashi; Radiana Dhewayani Antarianto

doi:10.3390/app11177887

Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy- 1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease

Blood ◽

10.1182/blood.v86.7.2842.2842 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2842-2848 ◽

Cited By ~ 189

Author(s):

M Korbling ◽

YO Huh ◽

A Durett ◽

N Mirza ◽

P Miller ◽

...

Keyword(s):

Body Weight ◽

Progenitor Cells ◽

Peripheral Blood ◽

Allogeneic Transplantation ◽

Hematopoietic Progenitor Cells ◽

Donor Blood ◽

Hematopoietic Progenitor ◽

Cd34 Cells ◽

The Mean ◽

Single Bone

Abstract Apheresis-derived hematopoietic progenitor cells have recently been used for allogeneic transplantation. Forty-one normal donors were studied to assess the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (12 micrograms/kg/d) on the peripheralization of hematopoietic progenitor cells and lymphoid subsets. The white blood cell, polymorphonuclear cell (PMNC), and lymphocyte concentrations at the peak of rhG-CSF effect in the donor's peripheral blood (PB) exceeded baseline by 6.4-, 8.0-, and 2.2-fold, respectively. Corresponding concentrations of PB CD34+ cells and primitive subsets such as CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells increased by 16.3-fold, 24.2-fold, and 23.2-fold, respectively in eight normal donors. The percentage of CD34+ Thy-1dim and CD34+ Thy- 1dim CD38- cells among CD34+ cells increased as well, suggesting an additional peripheralization effect of rhG-CSF on primitive CD34+ subsets. The preapheresis PB CD34+ and CD34+ Thy-1dim cell concentrations were predictive of their corresponding apheresis yield per liter of donor blood processed PB lymphoid subsets were not significantly affected by rhG-CSF treatment. The mean apheresis-derived yield of CD34+, CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 x 10(4) (n = 41), 27.2 x 10(4) (n = 10), and 1.9 x 10(4) (n = 10), respectively. As compared with 43 single bone marrow (BM) harvest, the CD34+ cell yield of peripheral blood progenitor cell allografts of 41 normal donors exceeded that of BM allografts by 3.7- fold and that of lymphoid subsets by 16.1-fold (CD3+), 13.3-fold (CD4+), 27.4-fold (CD8+), 11.0-fold (CD19+), and 19.4-fold (CD56+CD3-). All PBPC allografts were cryopreserved before transplantation. The mean recovery of CD34+ cells after freezing, thawing, and washing out dimethylsulfoxide was 86.6% (n = 31) and the recovery of lymphoid subsets was 115.5% (CD3+), 121.4% (CD4+), 105.6% (CD8+), 118.1% (CD19+), and 102.4% (CD56+CD3-). All donors were related to patients: 39 sibling-to-sibling, 1 parent-to-child, and 1 child-to-parent transplant. Thirty-eight transplants were HLA fully identical, two transplants differed in one and two antigens. Engraftment occurred in 38 recipients; two patients died too early to be evaluated, and one patient did not engraft. The lowest CD34+ cell dose transplanted and resulting in complete and sustained engraftment was 2.5 x 10(6)/kg of recipient body weight.(ABSTRACT TRUNCATED AT 400 WORDS)

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Encouraging Outcomes in African-American Patients with Use of Post-Transplant Cyclophosphamide after HLA Mismatched Hematopoietic Stem Cell Transplant

Blood ◽

10.1182/blood.v126.23.3228.3228 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3228-3228

Author(s):

David H. Aggen ◽

Kendra Mellert ◽

Divaya Bhutani ◽

Lois Ayash ◽

Lawrence G. Lum ◽

...

Keyword(s):

Overall Survival ◽

African American ◽

Chronic Gvhd ◽

Allogeneic Transplantation ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Post Transplant ◽

Gvhd Prophylaxis ◽

Unrelated Donors ◽

Equity Ownership

Abstract An inherent difficulty in performing allogeneic transplantation in patients (pts) of African American (AA) descent is finding suitably matched 8/8 unrelated donors. In addition for complicated reasons studies have shown that AA have an inferior survival compared to suitable matched patients from different ethnic groups undergoing allogeneic transplantation. Post transplant cyclophosphamide (PTCy) administered on day + 3 and +4 given in combination with tacrolimus and mycophenolate mofetil (MMF) allows for the safe use of more mismatched transplants, including haploidentical transplants. This has created a new approach for patients who lack suitable donors and may allow more AA pts to proceed to transplant. At our institution, we have used PTCy as graft-versus-host-disease (GVHD) prophylaxis in patients undergoing reduced intensity conditioning (RIC) hematopoietic stem cell transplant (HSCT) from mismatched related and unrelated donors, and this group consisted largely of high-risk AA pts. We retrospectively reviewed 16 pts who underwent HLA mismatched HSCT (5/10 to 8/10 HLA matches) at our institution between July 2012 and 2015. All patients received RIC HSCT with PTCy (days +3 and +4) along with tacrolimus and MMF for GVHD prophylaxis. Patients did not routinely receive growth factor support. Primary endpoints included time to neutrophil and platelet engraftment, length of hospital stay, and rates of acute and chronic GVHD. Secondary outcomes included time to relapse, transplant related mortality, and overall survival. Table 1 shows the demographics of the 16 patients who were transplanted. The median age of the patients was 57 years (range: 25-72). Ten pts were of AA descent. Fifteen donors were haploidentical family donors; 6 were 5/10 matches, 4 were 6/10 matches, and 5 were 7/10 matches. One patient received a 8/10 unrelated donor transplant. Five patients had failed previous transplants which included 3 allogeneic and 2 autologous HSCT. Based on published risk stratification (Armand et al. Blood, 2012) of the remaining 11 pts, 7 pts were high risk and 4 were intermediate risk. Neutrophil engraftment (first of 3 days when ANC was ³500 cells/mm3) occurred a median of 19 days post transplant (range:12-22 days); Platelet engraftment (³20,000/µL without platelet transfusion) occurred a median of 21.5 days post-transplant (range: 14-37 days). Median length of hospitalization was 26 days (range: 22-81 days). Two pts failed to engraft after first HSCT. One of these pts had high levels of anti-HLA antibodies directed against her donor. Both pts went on to a second haploidentical transplant and engrafted their neutrophil counts at 13 days and 21 days; respectively. Grade II acute GVHD occurred in 6/16 patients. No patient developed grade III-IV aGVHD. Chronic GVHD was observed in 8/16 patients with severe chronic GVHD seen in one patient. With a median follow up of 160 days (range: 89-778 days) the Kaplan-Meier estimates of overall survival at one year were 81.3% for all transplanted pts (Figure 1) and 80% for the AA pts (Figure 2). Three pts died; 1 patient from an invasive fungal infection and 2 pts from relapse. One additional patient relapsed but continues on treatment. Our experience suggests that the use of PTCy permits HLA mismatched transplantation, with overall survival of > 80% and acceptable rates of acute and chronic GVHD. Moreover, the severity of acute GVHD in patients who received PTCy post-transplant was limited, with no reported acute grade III-IV GVHD. In addition, the early outcomes of HLA mismatched transplantation in 10 African-American patients, a population that is often underrepresented in the donor registry, suggests that this approach may preferentially benefit AA pts from an expanded donor pool derived from utilization of partially HLA-matched donors. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Lum: Karyopharm Therapeutics Inc: Equity Ownership; Transtarget.Inc: Equity Ownership. Deol:Bristol meyer squibb: Research Funding.

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Identification of Highly Clonal Cells in CD34+ and Unselected Bone Marrow Samples From Patients with Myelodysplastic Syndrome Using a Sensitive Quantitative PCR Approach.

Blood ◽

10.1182/blood.v114.22.1760.1760 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1760-1760

Author(s):

Maximilian Mossner ◽

Daniel Nowak ◽

Ouidad Benlasfer ◽

Jana Reins ◽

Olaf Joachim Hopfer ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Progenitor Cells ◽

Quantitative Pcr ◽

X Chromosome ◽

Hematologic Malignancies ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 1760 Poster Board I-786 Myelodysplastic syndromes (MDS) are clonal hematologic malignancies with molecular defects most probably arising in the hematopoietic stem or progenitor compartment. However, due to a frequent lack of trackable cytogenetic aberrations in a large proportion of MDS patients the capability to monitor the manifestation and origin of malignant MDS clones remains limited. To elucidate clonal dominance in a given cell population, the analysis of skewed X-chromosome inactivation patterns in females, based on the methylation analysis of X-chromosomal HUMARA alleles has been used widely. However, this method has several technical and biological drawbacks as methylation changes can be induced with increasing age leading to inaccuracy of the method in this context. Recently, the application of a quantitative PCR-based method to accurately detect single nucleotide polymorphism (SNP) allele-specific RNA transcription from the X-chromosome (Swierczek et al, Blood, 2008) has shown robust results for reliable calculation of X-chromosome allelic ratios. In our study we employed this method to assess clonality in CD34+ selected and unselected bone marrow cells derived from MDS patients and provide evidence for distinct clonal manifestations of MDS clones in hematopoietic progenitor cells of all analysed MDS samples as compared to healthy controls. Bone marrow (BM) cells were obtained from patients with MDS (IPSS-low/int-1-risk n=9, IPSS-int-2/high-risk n=9) after informed consent. Immunomagnetic selection of CD34+ cells was performed from the BM samples of MDS patients (IPSS-low/int-1-risk n=8, IPSS-int-2/high-risk n=10) and age related healthy donors (n=6) served as controls for normalization. Genomic DNA SNP genotyping using Taqman SNP Genotyping Assays (Applied Biosystems, Foster City, CA) was carried out in order to screen for informative clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS and MPP1. RNA transcripts from different alleles were quantified using SNP/allele-specific primers in a Taqman based quantitative PCR approach. Individual allelic ratios were calculated as previously described. Clonality values were assigned to 0 % according to an allelic ratio of 50/50 (polyclonal) up to 100 % for a ratio of 100/0 (clonal). All clonality values are presented as mean. Our analyses revealed a remarkably elevated proportion of clonal cells in all purified CD34+ cells from MDS low/int-1-risk (88 %) and MDS int-2/high-risk patients (98 %) compared to the cells from healthy donors (16 %, p<0.001). The degree of clonality in unselected BM samples was similarly increased in MDS low/int-1-risk (74 %) and MDS int-2/high-risk specimen (87 %, both p<0.001 as compared to controls). However, whereas all purified CD34+ samples from MDS patients appeared to be highly clonal, 2 of 9 (22 %) of the MDS low/int-1-risk samples exhibited distinctively lower clonality in unselected BM cells with values comparable to the healthy control group. Furthermore, we observed nearly identical high clonality values in 12 paired BM/CD34+ MDS samples, except for 1 of 6 MDS low/int-1-risk samples with significantly lower clonality in the unselected bone marrow as compared to purified CD34+ cells of the same patient. Our observation of specific clonality in both unselected bone marrow and purified CD34+ cells of MDS patients as compared to healthy controls underlines the proliferative manifestation of malignant MDS clones even in early hematopoietic progenitor cells. Furthermore, the high degree of clonality in all purified CD34+ cells suggests a clonal involvement of not only myeloid but also lymphoid lineages. Interestingly, we also identified 2 patients harboring polyclonal cells in the unselected bone marrow. In these cases differentiating cells in the bone marrow may be sustained by residual healthy hematopoietic progenitor cells. The determination whether patients with this constellation have a different clinical prognosis from patients with clonality of the complete bone marrow may be interesting to pursue. In summary, determination of clonality levels in distinct cell populations of hematologic malignancies using quantitative PCR appears to be highly suitable for monitoring the manifestation and origin of a malignant hematopoietic stem/precursor cell. Disclosures No relevant conflicts of interest to declare.

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Expression of Complex Junction Proteins in Hematopoietic Progenitor Cells.

Blood ◽

10.1182/blood.v104.11.1282.1282 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1282-1282 ◽

Cited By ~ 1

Author(s):

Beate K. Straub ◽

Volker Eckstein ◽

Christine Grund ◽

Katrin Miesala ◽

Kerstin Horsch ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Adherens Junctions ◽

Actin Binding ◽

Hematopoietic Progenitor Cells ◽

Bovine Bone ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Junction Protein

Abstract The interaction of hematopoietic stem cells with the microenvironment plays a pivotal role in regulating self-renewal and differentiation. N-cadherin-based adherens junctions have been reported to be involved in this process (Puch et al., 2001; Zhang et al., 2003). However, the molecular composition of such junctions in hematopoietic progenitor cells (HPC) remains to be defined as well as their function. CD34+ cells and CD34+/CD38− cells were isolated from human umbilical chord blood (CB). In immunoblotting, CD34+ cells were positive for the transmembrane proteins N- and E-cadherin, cadherin 11 (low amounts) and the cytoplasmic plaque proteins α- and β-catenin, protein p120ctn, vinculin, spectrin/fodrin and actin as markers for adherens junctions as well as for ezrin, moesin and drebrin, actin-binding proteins known to be enriched in cellular protrusions. Other classical cadherins such as VE-, P-, R-cadherin or cadherin-6 were negative. Moreover HPCs were positive for the intermediate filament cytoskeletal protein vimentin, the integral heparan sulphate proteoglycans syndecan-1 (CD138), syndecan-2 (fibroglycan), syndecan-3 (N-syndecan) and syndecan-4 (ryudocan, amphiglycan) and the gap junction protein connexin 43. CD34+/CD38− cells showed positive reactions with antibodies against vimentin, ezrin, vinculin, α- and β-catenin and with a pan cadherin antibody, with no obvious differences to CD34+/CD38+ and Kg1a cells. In parallel, we established single- and double-immunohistochemistry with antigen-retrieval-treated, formalin-fixed, paraffin-embedded bone marrow using peroxidase, alkaline phosphatase and glucose oxidase coupled secondary antibodies. The architecture of human, rat and bovine bone marrow tissue was analyzed with a panel of antibodies against components of adhering, gap and tight junctions. Immunohistochemical sections of bone marrow incubated with the various antibodies remarkably showed the presence of all these antigens in their natural microenvironment. Intercellular junctions of rat and bovine bone marrow were also observed with transmission electron microscopy. Our results show that HPC are equipped with components of cadherin-catenin-based adherens junctions. Immunoprecipitation and immunolocalisation experiments are underway to further elucidate the homo- and heterotypic intercellular junctional complexes between HPC and stromal cells in vitro and in vivo.

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CD34+ Hematopoietic Progenitor Cells Express CD 184 Antigen and to a Minor Extend MMP9.

Blood ◽

10.1182/blood.v110.11.4032.4032 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4032-4032

Author(s):

Jens Abendroth ◽

Hans J. Schmoll ◽

Hans-Heinrich Wolf

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Immune Defense ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Mmp9 Expression ◽

Cd34 Cells

Abstract For clinical purposes the CD34 surface antigen characterises hematopoietic progenitor or stem cells (HSC) and will be measured routinely for peripheral hematopoietic stem cell transplantation (PBSCT). Concerning homing mechanisms of HSC not only the number of CD34+ cells could be important. However, there are several other characteristic surface antigens indicating proliferation. For example, the cell surface receptor CD184 has been found on CD34+ HSC and seems to be necessary for stem cell development and migration. CD184 knock-out mice are known to present insufficient hemato- and lymphopoiesis. MMP9, a gelatinase cleaving collagen typ IV, expressed on HSC, is necessary for migration and probably also for HSC engraftment. In leukapheresis products used for PBSCT we were interested in CD184 and MMP9 protease expression on CD34+ cells and their impact on homing. Methods: 84 leukapheresis products for autologous stem cell transplantation were analysed in 65 patients. The patients suffered from oncologic (n= 45) and haematological (n=20) malignancies. There was no bone marrow infiltration found before starting leukapheresis. Expression of CD184 and MMP9 on CD34+ cells were analysed by flow cytometry. Additionally, the number of CD45+ cells was measured (BD Biosciences). Statistical analysis was run by SPSS using students t-test and Mann-Whitney-U test. Results: 44,7% (+/− 3,1%) of CD34+ cells presented the CD184+ antigen, but MMP9+ expression was found only in 5% (+/− 1,4%) of CD34+ HSC, respectively. Regarding the patients age, sex or underlying diseases no significant differences were seen. Discussion: High expression rate of CD184 antigen and MMP9 on CD34+ HSC could be favorable for engraftment and success of hematopoietic stem cell transplantation. Surprisingly, in our study on leukapheresis products less than half of CD34+cells were found to express CD184+ antigen, and only 5% presented MMP9 expression. As time to engraftment after PBSCT of the patients did not differ from mean values either antigen expression could develop on a further step of hematopoietic reconstitution or the patients presented an "alternative pathway" establishing hematopoiesis and immune defense mechanisms. Further studies on proliferation and differentiation mechanisms of HSC are requested.

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Rapid and Prolonged Mobilization of Human CD34+ Hematopoietic Stem Cells Following Intravenous (IV) Administration of Plerixafor

Blood ◽

10.1182/blood.v116.21.2261.2261 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2261-2261 ◽

Cited By ~ 3

Author(s):

Michael P. Rettig ◽

Sandra Lopez ◽

Kyle McFarland ◽

John F. DiPersio

Keyword(s):

Body Weight ◽

Failure Rate ◽

Phase Ii ◽

Phase Ii Trial ◽

Fold Increase ◽

Allogeneic Transplantation ◽

Hematopoietic Stem ◽

Cell Dose ◽

Healthy Donors ◽

Cd34 Cells

Abstract Abstract 2261 Background: Plerixafor (AMD3100) is a CXCR4 antagonist that was approved by the FDA for use in combination with G-CSF to mobilize hematopoietic stem and progenitor cells (HSPCs) in patients with non-Hodgkin's lymphoma and multiple myeloma. We have previously reported the use of plerixafor alone to mobilize HSPCs for HLA-matched allogeneic transplantation (Blood. 2008;112:990). In this trial, sibling donors were treated with plerixafor at a dose of 0.24 mg/kg by subcutaneous (SC) injection, followed 4 hours later by leukapheresis. To date, 15 of 45 donors (33%) required a second day of leukapheresis to collect the minimum cell dose of ≥ 2.0 × 106 CD34+ cells/kg actual recipient body weight that are required to proceed to transplantation. Based on preliminary data suggesting higher (2-fold) and earlier (1 hr vs. 3 hr) progenitor mobilization in mice after intravenous (IV) dosing of plerixafor, we amended our trial to test the safety and efficacy of IV plerixafor. Methods: In our Phase I trial, 21 healthy donors were initially mobilized with increasing doses of IV plerixafor (0.08, 0.16, 0.24, 0.32, 0.40 or 0.48 mg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single SC dose of 0.24 mg/kg plerixafor followed 4 hours later by leukapheresis. In our Phase II study, 28 sibling donors were treated with plerixafor at a dose of 0.32 mg/kg by IV injection, followed 4 hours later by leukapheresis. Successful mobilization was defined as a minimum leukapheresis yield of ≥ 2.0 × 106/kg CD34+ cell/kg actual recipient body weight. Results: Peak CD34+ cells/mm3 were observed 4 hours after IV dosing (vs. 6–9 hours after SC dosing) and donors given 0.24 mg/kg IV plerixafor, had significantly higher peak levels of CD34+ cells/mm3 compared to the same donors who received 0.24 mg/kg SC plerixafor. There was a clear dose-response relationship of IV plerixafor on mobilization of CD34+ HSPCs, with the 0.32 mg/kg dose yielding a maximum increase in circulating CD34+ cells of 27 CD34+ cells/mm3 at 4 hours after injection, representing a median eight-fold increase from baseline. Interestingly, the median CD34+ cells/mm3 in donors receiving 0.40 mg/kg (N=3) and 0.48 mg/kg (N=3) was 17 and 24 CD34+ cells/mm3, respectively, not significantly better than the 320 mcg/kg dose at the four hour time-point. We also noted that IV dosing (especially doses >0.24 mg/kg) resulted in prolonged mobilization of CD34+ cells such that levels approached 20 CD34+ cells/mm3 at 24 hours after IV dosing. Pharmacokinetic studies demonstrated that the Cmax of plerixafor following the 0.32 mg/kg IV dose remained below 1.0 μg/mL whereas the 0.40 and 0.48 mg/kg doses resulted in Cmax levels of 1.8–2.2 μg/mL. A total of 28 HLA-identical sibling donor/recipient pairs have been enrolled in the Phase II trial, with all donors mobilized using IV plerixafor at a dose 0.32 mg/kg. At 4 hours after IV plerixafor, the CD34+ cell count rose to a median of 16 CD34+ cells/mm3 (range, 4–46), representing a 6.5-fold increase. The efficacy of CD34+ cell collection following 0.32 mg/kg IV plerixafor was not evaluable in five donors due to incomplete apheresis collections or the extremely large size of one recipient (recipient >200 kg). Six of the remaining 23 donors (26%) did not achieve the minimum cell dose of 2 × 106 CD34+ cells/kg in a single 20 L leukapheresis procedure. This mobilization failure rate of 26% with 0.32 mg/kg IV plerixafor is similar to the failure rate of 33% we observed following administration of 0.24 mg/kg SC plerixafor to healthy donors. Four of the six patients who failed to collect ≥ 2.0 × 106 CD34+ cells/kg after the first mobilization and apheresis procedure reached goal following a second mobilization and collection procedure. No adverse events or acute grade 3 or 4 toxicities have been observed in any of the donors given IV plerixafor doses up to 0.48 mg/kg. We have transplanted 24 of the 28 patients entered in the phase II trial. All but one patient engrafted neutrophils and platelets promptly and only 2 of the 24 (8.3%) transplant recipients have developed clinical evidence of acute GVHD (minimum follow-up of 45 days); 1 had grade II and the other grade III. Summary: IV plerixafor increases the magnitude and duration of CD34+ mobilization compared to SC plerixafor in healthy donors. These observations suggest that IV plerixafor may be a more effective mobilization agent with a low side effect profile for allogeneic transplantation. Disclosures: Rettig: Genzyme Corp.: Consultancy, Honoraria. DiPersio: Genzyme: Honoraria.

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Microrna-143 Blocks ERK5 Signaling During Granulocytic Differentiation of Hematopoietic Stem Cells and Is Downregulated in AML

Blood ◽

10.1182/blood.v120.21.3516.3516 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3516-3516

Author(s):

Jens-Uwe Hartmann ◽

Daniela Braeuer-Hartmann ◽

Cindy Schödel ◽

Dennis Gerloff ◽

Christiane Katzerke ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hematopoietic Progenitor Cells ◽

Atra Treatment ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Mapk Pathways ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 3516 Mitogen-activated protein kinase (MAPK) pathways are a family of related and sometimes interconnected pathways and one of the most studied. Over the last years, extensive work has established that these proteins play a critical role in G-CSF mediated maturation of neutrophil granulocytes. Understanding the mechanisms by which the MAPK pathways are regulated represents an important area of investigation. MicroRNAs, a class of small non-coding RNAs, have been found to play an important role in the regulation of diverse cellular processes by binding to target mRNAs leading to their translational repression. Deregulation of certain microRNAs, thereby, may lead to disrupted signal pathways, such as MAPK-signaling, and to tumorigenesis. However, the role of microRNAs in hematopoietic differentiation and development of leukemia remains largely unknown. In this study we performed a global screen to identify microRNAs involved in G-CSF-regulated MAPK-pathways in primary human CD34+ hematopoietic progenitor cells. Here we found microRNA-143 (miR-143) to be frequently upregulated in G-CSF stimulated CD34+ cells with a strong correlation to CD15 expression. We could also show the granulopoietic association of miR-143 in several hematopoietic cell line models and acute myeloid leukemia (AML) patient samples. Especially, AML patient samples FAB M4 and M5, which show monocytic phenotypes, had a significant lower expression level of miR-143 compared to the AML FAB types M0, M1, M2, and M3. In general, miR-143 expression was shown to be downregulated in AML patient samples in comparison to normal CD34+ hematopoietic progenitor cells. Most interestingly, we show that miR-143 expression is upregulated in acute promyelocytic leukemia (APL) patients after ATRA treatment. By in silico prediction we found MAPK protein family members (eg. MAPK1, MAPK3 and MAPK7) as predicted targets of miR-143. Western blot analysis of AML patient samples and G-CSF stimulated CD34+ cells clearly show an inverse correlation of miR-143 and MAPK7 (ERK5) protein expression. Finally, by transient overexpression of miR-143 we could show a strong downregulation of ERK protein expression in NB4 cells. Our study suggest that miR-143 upregulation by G-CSF may be an important regulatory step for permitting neutrophil differentiation. MicroRNA-143 blocks ERK5 signaling in G-CSF-induced granulopoiesis of CD34+ hematopoietic stem cells, is downregulated in myelo-monocytic acute myeloid leukemia subtypes, and upregulated after ATRA treatment in APL patients. This information may prove useful for the understanding of conditions in which neutrophil proliferation/differentiation balancing is dysregulated, such as in myeloid leukemia and myelodysplastic disorders. Disclosures: No relevant conflicts of interest to declare.

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Prediction of Hematopoietic Stem Cell Yield after Mobilization with GCSF in Healthy Unrelated Donors

Blood ◽

10.1182/blood.v124.21.1128.1128 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1128-1128

Author(s):

Raphael Teipel ◽

Johannes Schetelig ◽

Michael Kramer ◽

Helmuth Schmidt ◽

Alexander H. Schmidt ◽

...

Keyword(s):

Stem Cell ◽

Monocyte Count ◽

High Likelihood ◽

Normal Range ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Cd34 Cells ◽

Unrelated Donors ◽

Cell Mobilization ◽

Stem Cell Collection

Abstract Introduction: The collection of hematopoietic stem cells from the peripheral blood in healthy donors has been established as a highly efficient method in clinical practice. Nevertheless, there are some donors who mobilize poorly despite adequate mobilization regimes. Several factors influencing the process of stem cell mobilization have previously been discussed in the literature. Methods: In total, the data of 7.216 unrelated donors who underwent GCSF-induced stem cell mobilization and collection in two German apheresis centers between July 1997 and August 2012 were retrospectively reviewed. We systematically analyzed more than 30 factors with potential influence on the mobilization process and established a statistically stable model in order to predict the mobilization efficacy and the harvest success in unrelated stem cell donors. Based on empirical data and standard values, we created three model donors of each gender with different donor profiles (favorable/average/unfavorable). In those model donors we calculated the corresponding likelihood of a successful stem cell harvest dependent on the recipient’s weight. Results: Overall, ten variables with high statistical significance were included in the prediction model. Body mass index, platelet count, absolute lymphocyte count and the relative monocyte count correlated positively with the CD34+ count in the peripheral blood after five days of GCSF use (p < 0.0001 in a multivariate analysis). In contrast, female sex, age, smoking, lactate dehydrogenase (ldh), relative monocyte count, and the relative large unstained cell count were associated negatively with the CD34+ count on day five (p < 0.0001 in a multivariate analysis). In order to predict the harvest success (collection of > 2 x 10^6 CD34+ cells/kg recipient weight after first apheresis) three different models were compared in a ROC-analysis. The first model was purely based on female sex and recipient weight. The second model additionally contained the predicted CD34+ counts. Finally, in the third model the actual observed CD34+ counts were included. By adding the predicted CD34+ counts to the simple model, a significant improvement of the predictability of a harvest success could be viewed although a considerable difference comparing these results to the model with the observed CD34+ counts remained (figure 1). Furthermore, the prediction of harvest success in model donors (figure 2) revealed that donors with a favorable donor profile (covariates set to the most favorable values within the normal range) showed a particular high likelihood for a successful harvest (100 % likelihood, irrespective to donor’s gender or recipient’s weight). The likelihood for a successful harvest in male donors with an average distribution of covariates (covariates set to the empirical mean value) was nearly 100% as well even in donations for heavy recipients. Contrary, the likelihood for an average female donor was high in normal weight recipients (97 %; 60 kg recipient) but decreased with rising recipient weight (78 %; 140 kg recipient). In donors with an unfavorable profile (covariates set to the worst values within the normal range), especially in females, the chance for a successful stem cell collection was poor even when donating for light recipients (54 % in males, 10 % in females; 60 kg recipient). Conclusions: In conclusion, multiple factors with influence on the CD34+ count after GCSF mobilization in healthy donors have been identified. With the prediction model a significant gain in the predictability of a harvest success could be achieved though a certain amount of unexplained variance still remains. Model donors with a favorable or average donor profile had a high likelihood for a successful stem cell collection. In donors with an unfavorable profile, especially in females, the chance for a harvest success was very low. Figure 1: Prediction of harvest success (> 2 x 10^6 CD34+ cells/kg recipient weight after 1st apheresis) Figure 1:. Prediction of harvest success (> 2 x 10^6 CD34+ cells/kg recipient weight after 1st apheresis) Abb.: AUC – area under the curve Figure 2: Probability of harvest success in model donors (> 2 x 10^6 CD34+ cells/kg recipient weight after 1st apheresis) Figure 2:. Probability of harvest success in model donors (> 2 x 10^6 CD34+ cells/kg recipient weight after 1st apheresis) Disclosures Schmidt: Cellex GmbH: Employment. Ehninger:Cellex GmbH: Equity Ownership. Off Label Use: G-CSF.

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Characterization of CD34+ peripheral blood cells from healthy adults mobilized by recombinant human granulocyte colony-stimulating factor

Blood ◽

10.1182/blood.v84.8.2795.2795 ◽

1994 ◽

Vol 84 (8) ◽

pp. 2795-2801 ◽

Cited By ~ 96

Author(s):

GE Tjonnfjord ◽

R Steen ◽

SA Evensen ◽

E Thorsby ◽

T Egeland

Keyword(s):

Peripheral Blood ◽

Allogeneic Transplantation ◽

Healthy Adults ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Hematopoietic Stem ◽

Phenotypic Profile ◽

Cd34 Cells ◽

Stimulating Factor

Abstract Primed peripheral blood hematopoietic stem cells (PBSC) generate and sustain lymphohematopoiesis in myeloablated animals, and recent reports indicate that allogeneic transplantation using PBSC grafts may be feasible in humans. A major concern with the use of PBSC transplants is that permanent engraftment may be limited because of lack of sufficient numbers of primitive progenitor cells in the graft. In the present study, in vitro colony formation and immunophenotype of CD34+ cells in PB of healthy adults during short-term granulocyte colony-stimulating factor (G-CSF) administration were compared with that of CD34+ cells in normal bone marrow (BM). The number of CD34+ cells mobilized to PB peaked at day 4 or 5 of G-CSF administration. The phenotypic profile of CD34+ PB cells showed a substantial increase in the percentage of CD34+CD13+ and CD34+CD33+ cells (myeloid progenitors) and a corresponding decrease in the percentage of CD34+CD10+ and CD34+CD19+ cells (B lymphoid progenitors) compared with CD34+ BM cells. The other subsets studied, including CD34+CD38- and CD34+HLA-DR- cells, were present in both compartments in similar proportions. Furthermore, primed CD34+ PB cells were enriched for colony-forming cells (CFC) and displayed an increased clonogenicity when compared with their counterparts in BM. A comparison between a postulated PBSC graft and an average BM graft is presented, showing that such PBSC grafts will be enriched for CD34+ cells as a whole, CD34+CD33+ cells, and colony- forming cells (CFC), factors which have been shown to correlate to acceleration of hematologic reconstitution and reduction in requirements for supportive care in autografting. Hence, we predict that allogeneic transplantation using G-CSF-primed PBSC grafts will result in a more rapid hematologic reconstitution after myeloablative conditioning than BM grafting. The question of whether PBSC allografting will result in permanent engraftment and clinical benefits as observed in autografting has to be determined in prospective clinical studies.

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HLA-mismatched unrelated donors are a viable alternate graft source for allogeneic transplantation following alemtuzumab-based reduced-intensity conditioning

Blood ◽

10.1182/blood-2010-01-265413 ◽

2010 ◽

Vol 115 (25) ◽

pp. 5147-5153 ◽

Cited By ~ 48

Author(s):

Adam J. Mead ◽

Kirsty J. Thomson ◽

Emma C. Morris ◽

Sajir Mohamedbhai ◽

Shari Denovan ◽

...

Keyword(s):

Human Leukocyte ◽

Allogeneic Transplantation ◽

Reduced Intensity Conditioning ◽

Hematopoietic Stem ◽

Leukocyte Antigen ◽

Unrelated Donors ◽

Uniform Conditioning ◽

The Impact ◽

Reduced Intensity ◽

Related Mortality

Abstract The impact of human leukocyte antigen (HLA) mismatch after reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (RIT) using unrelated donors (UD) is unclear, and may be modulated by T-cell depletion. We therefore examined outcomes of 157 consecutive patients undergoing RIT after uniform conditioning with fludarabine, melphalan, and alemtuzumab (FMC). Donors were 10/10 HLA-matched (MUDs, n = 107) and 6 to 9/10 HLA-matched (MMUDs, n = 50), with no significant differences in baseline characteristics other than increased cytomegalovirus seropositivity in MMUDs. Rates of durable engraftment were high. Graft failure rates (persistent cytopenias with donor chimerism) were similar (8% vs 3%, P = .21), though rejection (recipient chimerism) was more frequent in MMUDs (8% vs 0%, P < .01). There were no significant differences between donors in the incidences of acute graft-versus-host disease (GVHD; 20% vs 22% grade 2-4, respectively, P = .83), chronic extensive GVHD (3-year cumulative incidence [CI] 23% vs 24%, P = .56), or treatment-related mortality (1-year CI 27% vs 27%, P = .96). Furthermore, there was no difference in 3-year overall survival (OS; 53% vs 49%, P = .44). Mismatch occurred at the antigenic level in 40 cases. The outcome in these cases did not differ significantly from the rest of the cohort. We conclude that RIT using HLA-mismatched grafts is a viable option using FMC conditioning.

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