Hematopoietic Stem Cell Yield Research Articles

The effect of plerixafor on autologous stem cell mobilization, cell viability, and apheresis challenges

Abstract Objective The aim of this study was to determine the efficacy of plerixafor for hematopoietic stem cell (HSC) mobilization prior to autologous stem cell transplantation (aSCT) for patients with multiple myeloma (MM) and various lymphomas, using an oncologist-guided HSC collection goal and markers of cell viability. Methods A retrospective chart review of all aSCT patients at Yale New Haven Hospital between 2017 and 2021 who met diagnostic criteria for MM, non-Hodgkin, or Hodgkin lymphoma (n = 382) was undertaken. Logistic regression evaluated plerixafor’s effect on meeting the individual’s HSC goal. The use of t-tests determined plerixafor’s relationship to HSC yield and analysis of variance testing assessed its effect on cell viability. Results Mobilization with granulocyte colony–stimulating factor (G-CSF) and plerixafor (odds ratio [OR] = 0.08; P < .05) relative to G-CSF alone was negatively associated with meeting the individual’s HSC goal. Diffuse large B-cell lymphoma in patients mobilized with plerixafor yielded fewer HSCs than those without plerixafor (t = –2.78; P = .03). Mobilization regimen (P = .13) had no association with HSC viability. Mobilization failure with plerixafor was rare but occurred in patients with multiple risk factors, including exposure to several rounds of HSC-affecting chemotherapy. Conclusion Plerixafor is effective across multiple diagnoses using an oncologist-driven HSC collection endpoint. Its association with mobilization failure is likely attributable to its use in patients predicted to be poor mobilizers.

A critical threshold of MCM10 is required to maintain genome stability during differentiation of induced pluripotent stem cells into natural killer cells.

Natural killer (NK) cell deficiency (NKD) is a rare disease in which NK cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Recently, a patient with NKD was identified carrying compound heterozygous variants of MCM10 (minichromosome maintenance protein 10), an essential gene required for DNA replication, that caused a significant decrease in the amount of functional MCM10. NKD in this patient presented as loss of functionally mature late-stage NK cells. To understand how MCM10 deficiency affects NK cell development, we generated MCM10 heterozygous (MCM10+/-) induced pluripotent stem cell (iPSC) lines. Analyses of these cell lines demonstrated that MCM10 was haploinsufficient, similar to results in other human cell lines. Reduced levels of MCM10 in mutant iPSCs was associated with impaired clonogenic survival and increased genomic instability, including micronuclei formation and telomere erosion. The severity of these phenotypes correlated with the extent of MCM10 depletion. Significantly, MCM10+/- iPSCs displayed defects in NK cell differentiation, exhibiting reduced yields of hematopoietic stem cells (HSCs). Although MCM10+/- HSCs were able to give rise to lymphoid progenitors, these did not generate mature NK cells. The lack of mature NK cells coincided with telomere erosion, suggesting that NKD caused by these MCM10 variants arose from the accumulation of genomic instability including degradation of chromosome ends.

A prospective, multicenter study on hematopoietic stemcell mobilization with cyclophosphamide plus granulocyte colony-stimulating factor and 'on-demand' plerixafor in multiple myeloma patients treated with novel agents.

High-dose melphalan plus autologous stem cell transplantation (ASCT) is a standard of care for transplant-eligible patients with newly diagnosed multiple myeloma (NDMM), and adequate hematopoietic stem cell (HSC) collection is crucial to ensure hematologic recovery after ASCT. In this prospective, observational study we evaluated HSC mobilization with granulocyte colony-stimulating factor (G-CSF), cyclophosphamide, and 'on-demand' plerixafor (in patients with <20×106 CD34+ cells/L after at least 4 days of G-CSF or failing to collect ≥1×106 CD34+ cells/kg after the first apheresis) in NDMM patients treated with novel agent-based induction therapy. The primary endpoint was the rate of poor mobilizers (patients collecting <2×106 CD34+ cells/kg or requiring plerixafor rescue to reach an adequate HSC harvest). Secondary endpoints included the rate of patients collecting ≥2×106 CD34+ cells/kg after plerixafor administration and the identification of factors predicting mobilization failure or plerixafor need. Overall, 301 patients (median age 60 years) were enrolled. Two hundred and eighty-seven of 301 (95%) and 274 of 301 (93%) patients collected ≥2×106 and ≥4×106 CD34+ cells/kg, respectively, with a median of 9.9×106 CD34+ cells/kg collected. Poor mobilizers were 48 of 301 (16%): 34 of 301 (11%) required plerixafor rescue, and 14 of 301 (5%) failed HSC collection regardless of plerixafor. Thirty-four of 38 (90%) patients receiving plerixafor collected ≥2×106 CD34+ cells/kg. Bone marrow plasmacytosis at diagnosis >60% (odds ratio [OR]=4.14), lenalidomide use (OR=4.45), and grade 3-4 hematologic toxicities during induction (OR=3.53) were independently associated with a higher risk of mobilization failure or plerixafor need. Cyclophosphamide plus G-CSF and 'on-demand' plerixafor is an effective strategy in NDMM patients treated with novel agents, resulting in a high rate of HSC collection and high HSC yield (clinicaltrials gov. identifier: NCT03406091).

Outcomes of patients after mobilization failure of hematopoietic stem cells for autologous stem cell transplantation

IntroductionAutologous hematopoietic stem cell transplantation (ASCT) is the long-term consolidation treatment for various hematological malignancies. The collection of hematopoietic stem cell yield is critical to successful ASCTs, but not always achieved due to hematopoietic stem cell mobilization failure (HSCMF). Details regarding the cell collection and outcomes of those who fail mobilization are still lacking. Therefore, this study aimed to yield data on clinical outcomes and cellular products after HSCMF. MethodsRetrospective, unicentric study assessing clinical outcomes and characteristics of collected progenitor cells. The data were collected from patient databases. The results were reported in median, rates and percentages and absolute values. Patients older than 18 years of age at the time of mobilization and HSCMF were included. ResultsFive hundred ninety-nine patients underwent mobilization protocols. Thirty-five (5.8%) of them failed in the mobilization and fourteen (40%) died. Median time to death was eight months. Disease progression and infection were responsible for all deaths. Median relapse-free survival was 6.5 months (20 patients, 57%). Seven (20%) survivors were receiving salvage therapy and five (14%) were being followed clinically. Six (20.6%) participants underwent collection by apheresis, with insufficient cell collection. The median quantity of peripheral CD34+ cells in those patients was 10.5/mm3. The median CD34+ quantity collected was 0.86 × 106 CD34+ cells/kg. ConclusionsThe mobilization failure was associated with limited survival. Nonetheless, collected products offered perspectives for ex vivo expansion. Further studies should investigate the feasibility of expanding collected CD34+ cells to use as grafts for ASCT.

Prediction of mobilized hematopoietic stem cell yield in patients with multiple myeloma: Usefulness of whole-body MRI-derived indices.

High-dose chemotherapy followed by autologous stem cell transplant is the mainstay of treatment for multiple myeloma (MM). The purpose of this study was to evaluate the ability of MRI-derived indices to predict mobilized hematopoietic stem cell yield. In this exploratory pilot work, we retrospectively analyzed 38 mobilization procedures for MM. Successful mobilization procedure was defined as a total yield of >4.0×106 CD34+ cells/kg. Univariate and multivariate analyses were performed to identify factors with a significant effect on successful mobilization from among clinical characteristics including number of prior lines of therapy, period from diagnosis to harvest, type of monoclonal protein (M protein); and radiological characteristics including total diffusion volume (tDV), median apparent diffusion coefficient (ADC) of tDV, and mean fat fraction of bone marrow calculated by MRI. Univariate analyses showed that relatively poor mobilization was significantly associated with M protein of Bence-Jones type and with median ADC of tDV (P = 0.02 and P = 0.004, respectively). Multivariate analyses using these two indices showed that median ADC of tDV was a significant predictive factor for adequate mobilization (P = 0.01), with an area under the curve of 0.784 (cutoff value, 1.18×10-3 mm2/s; sensitivity, 72.7%; specificity, 87.5%). The present data indicate that median ADC of tDV is a predictive factor for relatively poor mobilization of hematopoietic stem cells in MM patients undergoing autologous stem cell transplant.

Addition of bortezomib to high-dose cyclophosphamide therapy as a conditioning regimen for autologous peripheral blood stem cell harvest leads to an increased yield of hematopoietic stem cells.

Peripheral blood stem cell harvest (PBSCH) is a crucial procedure for autologous stem cell transplantation in patients with multiple myeloma. We herein report a retrospective study to verify the usefulness of bortezomib and high-dose cyclophosphamide therapy (Bor-HDCY) as a conditioning regimen for PBSCH. Thirty-three patients were evaluated. The median age at the first apheresis was 61 (interquartile range, 53-64) years old, and 18 (54.5%) patients were male. Bor-HDCY was performed in 15 patients, and HDCY was performed in 18. In the patients who underwent Bor-HDCY, the CD34+ cell count at the first apheresis was significantly higher than in the others (P<0.01), and the total CD34+ cell count also tended to be high (P=0.0933). In terms of apheresis days, two-thirds of the patients who underwent HDCY had two-day apheresis, whereas most who underwent Bor-HDCY had one-day apheresis. According to univariate analysis, Bor-HDCY (P<0.01), VRd (Bor, lenalidomide, and dexamethasone) as induction therapy (P=0.0529), and ≥VGPR before PBSCH (P=0.0767) were factors associated with a higher CD34+ cell count at first apheresis. Although multivariate analysis showed that there were no independently significant factors influencing the CD34+ cell count at the first apheresis, the stepwise selection method revealed that only the Bor-HDCY regimen remained in the final model (P<0.005). Bor-HDCY may be a useful conditioning regimen for increasing the CD34+ cell yield.

Assessment of factors affecting mobilization and collection in autologous and allogeneic hematopoietic stem cell donors

Background & Aim Background Peripheral blood hematopoietic stem cells (PBSC) are preferred source of stem cells in the majority of autologous and allogeneic transplantations. Sufficient number of mobilized and collected hematopoietic stem cells (HSC) is needed for successful transplantation. The aim of our study was to analyze possible predictive factors that could influence hematopoietic stem cell yield. Methods, Results & Conclusion Methods This study was performed in the Institute for Transfusion Medicine of RM and the University Hematology Hospital. There were 30 allogeneic and 90 autologous donors that underwent mobilization and collection of PBSC. The association between the number of collected PBSC and possible predictive factors was analyzed using Spearman Rank Order Correlation. Results There were 226 apheresis, 182 in autologous donors (mean 2, range 1-3) and 44 apheresis in 30 allogeneic donors (mean 1.5, range 1-2). The mean number of collected MNC in autologous donors was 3.09 × 108/kg and 2.85 × 106/kg CD34+ cells, and 3.23 × 108/kg MNC and 3.20 × 106/kg CD34+ cells in allogeneic donors. Significantly larger number of MNC and CD34+ cells was collected with the WBC set. There was a statistically significant correlation between the total number of collected MNC in autologous donors and platelet count before mobilization, the number of cycles in one apheresis procedure, quantity of collected graft and the number of collected MNC and CD34+ cells on the first day of apheresis. There was a statistically significant correlation between the total number of collected MNC in allogeneic donors and platelet count before mobilization, the number of cycles in one apheresis procedure, quantity of collected graft and number of MNC on the first day of apheresis. There was a strong correlation between the number of collected MNC and CD34+ cells on the first harvest and the total number of collected MNC and CD34+ cells in poor mobilizers, and inverse correlation with the number of apheresis procedures. Donors who donated MNC ≤ 0.7 × 108/kg and/or ≤ 0.7 × 106/kg CD34+ cells on the first harvest (84.6%) were strong predictors of poor mobilizers. Conclusion Determining the proper level of laboratory parameters for initiating mobilization and apheresis procedure which is safe for donors and efficient in collection of PBSC is needed for optimization of these procedures, as well as for early intervention in poor mobilizers.

Current and future status of stem cell expansion.

Herein, we seek to describe the current and future role of ex-vivo expansion of cord blood hematopoietic stem cells. As this field is only in its infancy, there have been many challenges identified. Decreased number of stem cells contained in a cord blood unit and early differentiation of stem cells once expanded have been two overarching challenges faced by the field. Many recent techniques have focused on the properties of the microenvironment and targetable cellular pathways as novel approaches to circumvent these challenges. Novel discoveries have led to the development of approaches that will increase hematopoietic stem cell yield and will improve engraftment in patients receiving cord blood hematopoietic stem cell transplantation. As a result, patients receiving cord blood hematopoietic stem cell transplantationcontinue to have improved outcomes.

A novel association between relaxin receptor polymorphism and hematopoietic stem cell yield after mobilization.

Mobilization of hematopoietic stem cells (HSCs) from the bone marrow to the peripheral blood is a complex mechanism that involves adhesive and chemotactic interactions of HSCs as well as their bone marrow microenvironment. In addition to a number of non-genetic factors, genetic susceptibilities also contribute to the mobilization outcome. Identification of genetic factors associated with HSC yield is important to better understand the mechanism behind HSC mobilization. In the present study, we enrolled 148 Korean participants (56 healthy donors and 92 patients) undergoing HSC mobilization for allogeneic or autologous HSC transplantation. Among a total of 53 polymorphisms in 33 candidate genes, one polymorphism (rs11264422) in relaxin/insulin-like family peptide receptor 4 (RXFP4) gene was significantly associated with a higher HSC yield after mobilization in Koreans. However, in a set of 101 Europeans, no association was found between circulating CD34+ cell counts and rs11264422 genotype. Therefore, we suggest that the ethnic differences in subjects’ genetic background may be related to HSC mobilization. In conclusion, the relaxin—relaxin receptor axis may play an important role in HSC mobilization. We believe that the results of the current study could provide new insights for therapies that use relaxin and HSC populations, as well as a better understanding of HSC regulation and mobilization at the molecular level.

Can A Single Exercise Bout Increase The Yield Of Hematopoietic Stem Cells From Peripheral Blood?

Hematopoietic stem cell transplantation (HSCT) is a common treatment option for patients with hematological malignancies. HSCT donors receive twice-daily injections of mobilizing pharmaceutical agents for 4-5 days before beginning apheresis for an additional 3-4 days. Not only is this a costly and time consuming process, the pharmaceuticals used to mobilize stem cells have several undesirable side effects. A single bout of exercise increases stem cell counts in peripheral blood and may therefore serve as a suitable adjuvant for mobilizing stem cells in allogeneic donors and lessen the need for additional mobilizing agents. PURPOSE:To compare short duration high intensity exercise with long duration low intensity exercise in an effort to determine which exercise modality would mobilize the greatest number of stem cells. METHODS:11 Healthy runners completed two exercise sessions in randomized order: 90 minutes at 5% below VT1 (LONG) and 30 minutes at 15% above VT1 (SHORT). Blood samples were taken pre-exercise, mid-exercise (15 and 90 min into exercise), immediately post exercise, and into the recovery period (1-Hr, 2-Hr and 3-Hr Post). Total CD34+ stem cells in whole blood were enumerated by 4-colour flow cytometry. RESULTS:Compared to pre-exercise, SHORT yielded a greater post-exercise (mean ± SE) CD34+ cell mobilization than LONG (+6.7±1.96 vs. +0.32± 1.71, p=0.069). Peak CD34+ cell mobilization occurred immediately post exercise for SHORT and mid-exercise for LONG (+6.7±1.96 vs. +2.62 ±1.33, p=0.142). CONCLUSIONS: Short duration high intensity exercise elicits a larger mobilization of CD34+ cells than long duration exercise of lower intensity. Exercise may therefore serve as a suitable adjuvant to mobilize CD34+ cells in healthy donors, although further research is necessary to optimize the exercise protocols for the HSCT donor population.

The High Effect of Chemomobilization with High-Dose Etopside + Granulocyte-Colony Stimulating Factor in Autologous Hematopoietic Peripheral Blood Stem Cell Transplantation: A Single Center Experience.

Autologous hematopoietic stem cell transplantation (auto-HSCT) provides hematopoietic support after high-dose chemotherapy and is the standard of care for patients with multiple myeloma (MM), chemo sensitive relapsed high or intermediate grade non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL). However, yields of hematopoietic stem cells vary greatly between patients, and the optimal strategy to mobilize hematopoietic stem cells into peripheral blood for collection has not been defined yet. We investigated the efficacy and safety of chemo mobilization with an intermediate dose etoposide (VP-16; 200 mg/m2 on days 1-3) and granulocyte-colony stimulating factor (G-CSF)(5 µg/kg twice daily from day 4 through the final day of collection). We reviewed our institutional experience with 91 patients (71 MM, 12 HL, 8 NHL) mobilized with this regimen. VP-16 + G-CSF resulted in successful mobilization in 95.55% of the patients (on one patient stem cell collection with plerixafor was applied), including 76 patients (83.52%) whose stem cells were collected successfully in a single day. Collection was managed between min. D8 and max. D17. Patient age, gender, exposure to previous irradiation and chemotherapy, previous mobilization attempts, and disease characteristics were not considered during selection. Adverse effects of the regimen included supportive transfusions and fevers requiring hospitalization or intravenous antibiotics. VP-16 and G-CSF appears to be a safe and effective mobilization regimen for patients with multiple myeloma, non-Hodgkin’s lymphoma and Hodgkin’s lymphoma undergoing autologous stem cell transplantation, producing excellent stem cell yield with the majority of patients requiring 1 day of apheresis.

Circulating hematopoietic progenitors and CD34+ cells predicted successful hematopoietic stem cell harvest in myeloma and lymphoma patients: experiences from a single institution

BackgroundPrevious studies have shown that the numbers of both circulating hematopoietic progenitor cell (HPC) and CD34+ cell are positively correlated with CD34+ cell harvest yield. However, the minimal numbers of both circulating HPCs and CD34+ cells required for performing an efficient hematopoietic stem cell (HSC) harvest in lymphoma and myeloma patients have not been defined in our institution.Patients and methodsMedical records of 50 lymphoma and myeloma patients undergoing peripheral blood HSC harvest in our institution were retrospectively reviewed. The minimal and optimal HSC harvest yield required for the treatment was considered to be ≥2×106 CD34+ cells/kg and ≥5×106 CD34+ cells/kg, respectively.ResultsThe minimally required or optimal HSC yield obtained was not influenced by age (≥60 years), sex, underlying malignancies, disease status, multiple rounds of chemotherapy, or history of radiotherapy. The numbers of both circulating HPC and CD34+ cell were higher in patients with minimally required HSC yields (P=0.000 for HPC and P=0.000 for CD34+ cell) and also in patients with optimal HSC yields (P=0.011 for HPC and P=0.006 for CD34+ cell). The cell count cutoff for obtaining minimally required HSC harvest was determined to be 20/mm3 for HPCs and 10/mm3 for CD34+ cells. Furthermore, the cell count cutoff for obtaining optimal HSC harvest was determined to be 60/mm3 for HPCs and 35/mm3 for CD34+ cells.ConclusionA total of 60/mm3 of HPCs and 35/mm3 of CD34+ cells in peripheral blood predicted optimal HSC harvest in lymphoma and myeloma patients.

Prediction of hematopoietic stem cell yield after mobilization with granulocyte-colony-stimulating factor in healthy unrelated donors.

The collection of hematopoietic stem cells from the peripheral blood of healthy donors has been established as a highly efficient method. Nevertheless, some donors have a moderate or poor chance of harvest success with standard mobilization regimens. We retrospectively reviewed data from 7216 unrelated healthy donors, who underwent granulocyte-colony-stimulating factor mobilization and consecutive leukapheresis for allogeneic stem cell transplantation. We tested different donor variables of potential influence and established a statistical model for prediction of upfront mobilization capacity and harvest success. In addition, we calculated the likelihood of a successful harvest dependent on predicted preapheresis CD34+ count and recipient weight. Female sex, older age, smoking, elevated lactate dehydrogenase, higher relative lymphocyte count, and higher large unstained cell count at baseline were negatively correlated with the CD34+ cell count on Day +5 (p < 0.0001). In contrast, higher platelet count, higher body mass index, higher absolute lymphocyte count, and higher relative monocyte count at baseline showed a positive correlation with the CD34+ count on Day +5 (p < 0.0001). Using a model built on these factors, we could significantly improve the prediction of harvest success compared to a basic model. The model allows the identification of female donors who eventually have a significant risk of harvest failure if requested to donate for recipients with a high body weight.

A Retrospective Study on Safety, Efficacy and Cost of the Chemo-Mobilization for Patients Planned to Undergo Autologous Hematopoietic Stem Cell Transplant: The Secom Study

Introduction: Failure of hematopoietic stem cell (HSC) mobilization occurs in 5 to 30% patients planning for high dose chemotherapy and autologous HSC transplantation worldwide. It has adverse impact on patient outcomes and significantly increases health care burden. Data regarding HSC mobilization based on chemotherapy + granulocyte colony stimulating factor (G-CSF) in Asia are currently limited.Objective: The primary objective was to determine the safety and efficacy of HSC chemo-mobilization protocols in Asia for patients with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM) planning for autologous HSCT. The secondary objective was to provide an estimate of the cost of chemo-mobilization in Asian countriesStudy Design: This was a multicenter, multinational retrospective observational study. Patients with NHL or MM undergoing chemo-mobilization for planned autologous HSCT between 1 Jan 2009 to 31 Dec 2012 in five Asian countries including Thailand, Singapore, Malaysia, Hong Kong and Taiwan, were retrospectively included in the study. Patient demographics, disease diagnoses, previous treatment history as well as complete blood counts prior to chemo-mobilization and apheresis, were collected.Results: A total of 526 patients (male/female = 207/219) were analyzed (diagnoses: NHL=257; MM=269). 160 patients were recruited from Thailand, 98 from Malaysia, 161 from Taiwan, 59 from Singapore and 48 from HK. Median age for the overall group was 53 years (range: 15-82). The most common mobilization regimen was cyclophosphamide + G-CSF; 235 (87.4%) in MM and 72 (28%) in NHL patients. 448 (85.2%) patients had reached at least HSC yield of 2 x 106CD 34+ cells/kg in 1 or 2 apheresis days. During chemo-mobilization, 108 (20.5%) patients were without negative clinical events (grade 3/4 neutropenia, febrile neutropenia, prolonged hospitalization, bone pain, fever, gastrointestinal, line infections or others). Grade 3/4 neutropenia and febrile neutropenia occurred in 401 (76.2%) and 72 (13.7%) patients, respectively. Median number of apheresis sessions was 2 (range: 1-5). Median cost of mobilization was 6,200USD (9,000-48,000). The respective median costs in USD (range) was; 3,500 (1,500-28,500) in Thailand, 6,900 (2,300-29,600) in Malaysia, 7,700 (3,400-48,000) in Taiwan and 9,200 (900-45,400) in Singapore. 436 (82.9%) patients proceeded to receive high dose chemotherapy and HSCT but 94 (17.87%) patients did not, due to insufficient HSC yield (n=26, 4.94 %), progression of disease (n=18, 3.42 %), patient not fit/ withdrawal from study (n=19, 3.61 %), patient expiry (n=11, 2.09 %) and other reasons (n=20, 3.80 %).Conclusion: Cyclophosphamide and G-CSF is the most common mobilization regimen used in Asian countries. Current chemo mobilization regimens are associated with a satisfactory rate of successful stem cell collection but with a high rate of significant toxicity. More than three-quarter of patients suffered from grade 3/4 toxicity during chemo-mobilization, however, only 20% of them had febrile neutropenia. Variations exist in the cost of chemo-mobilization in Asia, both among and within countries. DisclosuresGoh:Novartis Pte Ltd: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceuticals Inc: Research Funding; Bristol-Myres Squibb: Membership on an entity’s Board of Directors or advisory committees; Gilead Sciences: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Hospira Inc: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

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

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. [Display omitted] Abb.: AUC – area under the curve [Display omitted] DisclosuresSchmidt:Cellex GmbH: Employment. Ehninger:Cellex GmbH: Equity Ownership. Off Label Use: G-CSF.

Optimization Of Stepwise Hematopoietic Differentiation Of Human iPSCs

Since their discovery in 2006, induced pluripotent stem cells (iPSCs) with their ES cell-like self-renewal and differentiation capability, are set to revolutionize the field of regenerative medicine. There is tremendous interest in the field of hematology for derivation of hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPCs) by in vitro differentiation of IPSCs. IPSCs can be differentiated into HSC/HPCs by coculture on feeder cells, such as OP9, or by using stepwise differentiation protocols on defined media. Neither approach produces high yields of HSCs or HPCs. With an intention to improve this, we systematically investigated various parameters for in vitrodifferentiation of iPSCs into HPCs.iPSCs were derived from human adult dermal fibroblasts by transduction with the Yamanaka retroviral vectors (encoding human Klf4, Oct3/4, Sox2 and cMyc) or by electroporation with the Yamanaka Epstein–Barr virus-based episomal plasmid vectors (encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA). One iPSC clone of each variety was then subjected to a stepwise differentiation protocol described by Niwa and coworkers [PLoSOne. (2011); 6(7):e22261] followed by hematopoietic colony forming (CFU) assays in MethoCult (STEMCELL Technologies, Vancouver, Canada). The original protocol calls for the use of Stemline II serum-free medium (Sigma, St. Louis, MO) supplemented with various growth factors/cytokines. We investigated the use of APEL medium described by Ng and coworkers [Nature Protocols. (2008); 3(5): 768] as a possible substitute for Stemline II. We also tested the effect of varying the number of colonies seeded in 6-well plates and the efficiency of hematopoietic differentiation after seeding iPSCs as single cells. The results, based on the number of hematopoietic colonies obtained in MethoCult following differentiation, showed that the APEL medium (>100 CFU/100,000 cells) was a superior substitute to the Stemline II medium (<10 CFU/100,000). When IPSCs were seeded as single cells, at initial densities of 10, 100 or 1,000 cells/cm2 in the presence of Y-27632 Rock inhibitor, only the cells at starting density higher than 1,000 per cm2survived but did not yield hematopoietic CFUs in MethoCult. When seeded as colony fragments, lower density of seeding in 6-well plates (< 20 colonies/well) was superior to higher density (>50 colonies/well) for obtaining HPCs. Other parameters that can affect differentiation, such as bone-morphopoietic protein (BMP) and O2 concentration, are being investigated.Figure A. Cellular markers detected at different time points of stepwise hematopoietic differentiation. B. CFUs in MethoCult. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Increasing Hematopoietic Stem Cell Yield to Develop Mice with Human Immune Systems

Hematopoietic stem cells (HSCs) are unique in their capacity to give rise to all mature cells of the immune system. For years, HSC transplantation has been used for treatment of genetic and neoplastic diseases of the hematopoietic and immune systems. The sourcing of HSCs from human umbilical cord blood has salient advantages over isolation from mobilized peripheral blood. However, poor sample yield has prompted development of methodologies to expand HSCs ex vivo. Cytokines, trophic factors, and small molecules have been variously used to promote survival and proliferation of HSCs in culture, whilst strategies to lower the concentration of inhibitors in the culture media have recently been applied to promote HSC expansion. In this paper, we outline strategies to expand HSCs in vitro, and to improve engraftment and reconstitution of human immune systems in immunocompromised mice. To the extent that these “humanized” mice are representative of the endogenous human immune system, they will be invaluable tools for both basic science and translational medicine.

Effects of preeclampsia on the yield of hematopoietic stem cells obtained from umbilical cord blood at delivery

To compare the numbers of cord blood CD34(+) hematopoietic stem cells (HSC) between preeclampsia (PE) and control (non-PE) subjects and to determine the factors that may influence this observation. Umbilical cord blood was collected from 28 PE and 19 non-PE subjects. Nucleated and CD34(+) cell counts were derived using the Trucount tube-based stem cell enumeration kit on BD FACSCalibur. The cord blood volume, nucleated and CD34(+) cell counts were significantly reduced in PE subjects compared to non-PE subjects. Among the PE subjects, systolic and diastolic blood pressure demonstrated a negative correlation with total nucleated and CD34(+) cell counts. Gestational age at delivery influenced cord blood volume and nucleated cell counts, but not CD34(+) cell counts. Birth weight and placental weight correlated strongly with cord blood volume, and nucleated and CD34(+) cell counts. There were no correlations observed between cord blood parameters and maternal age, maternal white cell count, gravidity, route of delivery or neonatal gender among PE subjects. Preeclampsia has a negative impact on the yield of HSC obtained from cord blood at delivery. Maternal blood pressure, neonatal birth weight and placental weight are important factors influencing the numbers of cord blood HSC. These findings should be taken into consideration when selecting cord blood units from mothers with PE for banking. Selecting the heaviest term neonate might improve the yield of cord blood HSC obtained from PE mothers.

Temporal Changes in Plerixafor Administration Do Not Impact Hematopoietic Stem Cell Mobilization Efficacy: Results of a Prospective Clinical Trial

Abstract 2988 Objectives:Plerixafor (AMD3100, Mozobil) with filgrastim (G-CSF, Neupogen) is approved for hematopoietic stem cell (HSC) mobilization in patients with non-Hodgkin Lymphoma and multiple myeloma (MM). Plerixafor pharmacokinetics (PK) and pharmacodynamics (PD) are well described, with linear, dose-dependent PK following subcutaneous (SC) administration, peak concentrations 30–60 mins post-injection and an elimination half-life (t1/2) of 5.3 hr. In pharmacodynamic studies of plerixafor in conjunction with filgrastim in healthy volunteers, peak CD34+ cell counts occur 10–14 hours following administration, however, data is limited in the 14–24 hr timeframe. Plerixafor labeling requires SC dosing approximately 11 hours prior to apheresis, which translates into dosing 10 :00 PM the night before apheresis, and 54% of MM patients collect ≥ 6 × 106 CD34+ cells/kg following a single apheresis procedure. The current regimen is inconvenient for patients and requires additional health care resources. Based on PK and PD, we hypothesized that plerixafor given at 3 :00 PM (17 hr prior to apheresis) would yield equivalent CD34+ HSC yield to 10 :00 PM dosing in MM patients. Methods:In a Simon’s two-stage design, we enrolled MM patients undergoing cytokine-only HSC mobilization. All subjects received filgrastim 7.5 mcg/kg SC BID for 4 days followed by plerixafor (0.24 mg/kg SC daily) for up to 4 days beginning at 3 :00 PM the day prior to the first day of a 24-liter apheresis procedure at 8 :00 AM. Target CD34+ HSC collection for stem cell transplant (SCT) was ≥ 10 × 106 CD34+ cells/kg. Blood samples for CD34+ fluorescence-activated cell sorting analysis were collected prior to the first plerixafor dose and at 1, 3, and 17 ± 1 hr, then daily prior to apheresis as needed. Results:Thirty patients (17 female, median age 59 years [range 44–70]) were evaluable; 27 received 1 pre-mobilization regimen (RVD n=20, VTD n=2, VD n=2, V/PLD/D n=1, VT n=1, RD n=1) for a median of 4 (1–6) cycles. Three received 2 regimens [CMF × 6 (breast cancer), then VTD × 5; RD × 4, then RVD × 4; and V/PLD × 1 with maintenance R]. Six patients received prior radiation. Mean (± SD) CD34+ cell counts in peripheral blood pre-plerixafor and 1, 3, and 17 hr post-first dose increased through the dosing interval (Figure). Twenty-two (73%) patients collected target cell numbers in 1 day of apheresis, 7 (23%) in 2 days, and 1 (3%) in 3 days. Twenty-seven (90%) patients collected ≥ 6 × 106 CD34+ cells/kg in 1 day. Institutional data with filgrastim 7.5 mcg/kg SC BID for 4 days alone in MM in 22 subjects showed a day 1 collection of ≥ 10 × 106 CD34+ cells/kg in 18% of patients (Renfroe H, et al. Transfusion Feb 2011). Adverse events were generally mild and consistent with known side effects of the combination [gastrointestinal disorders (diarrhea, nausea) and injection site reactions]. To date, 16 (53%) patients have proceeded to autologous SCT with melphalan conditioning and all patients have engrafted, with median time to an ANC ≥ 500/mm3 of 13 (range 11–15) days and platelets ≥ 20, 000/mm3 of 16 (range 11–21) days. Conclusion:This is the first prospective trial demonstrating the safety and efficacy of plerixafor given 17 hr prior to apheresis. Pharmacodynamic data showed the peripheral blood CD34+ cell population increased throughout the dosing interval, with a 4.6-fold increase over pre-plerixafor counts at 17 hr. Comparison with historical institutional controls and published data suggests this regimen yields at least equivalent, if not superior, collection rates with one apheresis procedure. [Display omitted] Disclosures:Flowers:Genentech/Roche (unpaid): Consultancy; Celgene: Consultancy; Millennium/Takeda: Research Funding; Wyeth: Research Funding; Novartis: Research Funding.

Factors affecting autologous peripheral blood hematopoietic stem cell collections by large-volume leukapheresis: a single center experience.

To evaluate factors affecting peripheral blood hematopoietic stem cell yield in patients undergoing large-volume leukapheresis for autologous peripheral blood stem cell collection. Data from 304 consecutive autologous peripheral blood stem cell donors mobilized with hematopoietic growth factor (usually G-CSF), associated or not with chemotherapy, at Hospital Israelita Albert Einstein between February 1999 and June 2010 were retrospectively analyzed. The objective was to obtain at least 2 × 106 CD34+ cells/kg of body weight. Pre-mobilization factors analyzed included patient's age, gender and diagnosis. Post mobilization parameters evaluated were pre-apheresis peripheral white blood cell count, immature circulating cell count, mononuclear cell count, peripheral blood CD34+ cell count, platelet count, and hemoglobin level. The effect of pre and post-mobilization factors on hematopoietic stem cell collection yield was investigated using logistic regression analysis (univariate and multivariate approaches). Pre-mobilization factors correlating to poor CD34 + cell yield in univariate analysis were acute myeloid leukemia (p = 0.017) and other hematological diseases (p = 0.023). Significant post-mobilization factors included peripheral blood immature circulating cells (p = 0.001), granulocytes (p = 0.002), hemoglobin level (p = 0.016), and CD34+ cell concentration (p < 0.001) in the first harvesting day. However, according to multivariate analysis, peripheral blood CD34+ cell content (p < 0.001) was the only independent factor that significantly correlated to poor hematopoietic stem cell yield. In this study, peripheral blood CD34+ cell concentration was the only factor significantly correlated to yield in patients submitted to for autologous collection.