Stem Cell Yield Research Articles

Impact of Melphalan-Based Chemotherapy on Stem Cell Collection in Patients with Light Chain Amyloidosis

Introduction: Melphalan based chemotherapy is commonly used for treatment of light chain amyloidosis (AL). Patients with AL often receive chemotherapy before autologous stem cell transplantation (ASCT) if they have high plasma cell burden or while awaiting ASCT. Melphalan is an alkylator and can affect bone marrow stem cells. Limited data is available on the effect of melphalan on stem cell mobilization in patients with amyloidosis. This study aims to identify the impact of melphalan therapy on collection of stem cells and ASCT in amyloidosis.Methods: All patients with AL seen at our institution within 90 days of diagnosis over a 10-year period (2006 to 2015) who underwent stem cell harvest were identified from an institutional database. Data pertaining to demographics, diagnosis, treatment, stem cell harvest and ASCT was extracted from the electronic medical records. Analysis was carried out by chi-square and Fisher's exact test for categorical variables and Kruskal-Wallis and Wilcoxon rank sum test for ordinal and continuous variables.Results: Three hundred and seventy two patients with AL who met the inclusion criteria were identified, of whom 10% (n=38) received melphalan based chemotherapy prior to harvesting, 28.5% (n=106) received non-melphalan based chemotherapy and 61.3% (n=228) received no chemotherapy prior to stem cell collection. Bortezomib based regimens were the most common (78%, n=83) non-melphalan based chemotherapy.All three groups were similar in terms of median age at diagnosis (59.1 years), median age at collection (59.4 years), gender distribution (59% males, n=221) and type of involved free light chain (FLC), with lambda being more common (72.2%, n=268). Patients who received melphalan-based chemotherapy had more cardiac (73.8% vs. 45.2% vs. 46.4%, p=0.005) and renal (84.2% vs. 50.9% vs. 68%, p=0.0002) involvement compared to other chemotherapy and no chemotherapy groups, respectively. In contrast, patients who received non-melphalan based chemotherapy had higher plasma cell burden (15% vs. 6% vs. 10%, p< 0.0001) and greater difference between involved and uninvolved FLC (44.2 mg/dL vs. 13.3 mg/dL vs. 13.2 mg/dL, p< 0.0001) compared to melphalan and no chemotherapy, respectively.Median duration of melphalan based chemotherapy was shorter at 54 days (34.5 to 79.5) or estimated 2 cycles compared to 101 days (60 to 135.5) or estimated 4 cycles (p=0.0019). Despite shorter duration of chemotherapy, total stem cell yield (million CD34/kg) was lower in patients who received melphalan based chemotherapy (5.54) compared to non-melphalan based chemotherapy (8.14) or no prior chemotherapy (7.94); p<0.0001. Similarly, day one stem cell yield (million CD34/kg) was the lowest in the melphalan group (2.71), followed by other chemotherapy group (3.63) and highest in no chemotherapy group (4.84); p<0.0001. This trend persisted for average stem cell yield per collection as illustrated in table 1.Filgrastrim (GCSF) alone was the most common mobilizing agent. However, patients with any chemotherapy prior to harvesting had higher utilization of plerixafor; 26.3% (n=10) in the melphalan group and 39.6% (n=42) in the non-melphalan group compared to 11.6% (n=27) if no prior chemotherapy (p<0.0001). However, no statistically significant difference was seen for melphalan vs. non-melphalan chemotherapy groups (p=0.44).In patients who underwent ASCT (85%, n=315), median stem cell dose (million CD34/kg) was different in the melphalan (3.66), non-melphalan (4.2) and no chemotherapy groups (4.44) (p=0.047), though the difference was not statistically significant amongst the 2 chemotherapy groups (p=0.34). There was also no difference in time to engraftment (table 1).Conclusions: Melphalan based chemotherapy, even if used for a short duration of time, significantly decreases both total stem cell yield and the yield on day one. It therefore has the potential to add to resource utilization with more collections needed. As much as possible, limited cycles of melphalan based chemotherapy or non-melphalan based treatment should be utilized in patients who are transplant eligible. [Display omitted] DisclosuresDispenzieri:GSK: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Jannsen: Research Funding; Celgene: Research Funding; pfizer: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kapoor:Amgen: Research Funding; Takeda: Research Funding; Celgene: Research Funding. Kumar:Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Glycomimetics: Consultancy; BMS: Consultancy; Sanofi: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Kesios: Consultancy.

A Pilot Study of Eltrombopag Plus G-CSF for Human CD34+ Cell Mobilization in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplant

Background: Administration of recombinant human thrombopoietin (rhTPO) with G-CSF for stem cell mobilization is associated with high CD34+ stem cell yield, rapid neutrophil recovery following autologus transplantation (ASCT), and decreased red blood cell (rbc) and platelet (plt) transfusions (Solomo, G. et al. Blood 1999). However, clinical development of rhTPO was complicated by the formation of neutralizing anti-TPO antibodies (Li, J. et al. Blood 2001), prompting discontinuation of further clinical development of recombinant TPO. Eltrombopag (Elt) is an orally bioavailable small molecule thrombopoietin receptor (TPO-R) agonist approved by the FDA for treatment of chronic immune thrombocytopenic purpura (ITP). In vitro studies have demonstrated that Elt promotes megakaryocyte proliferation and differentiation of CD34+ bone marrow progenitor cells (Erickson-Miller CL Stem Cells 2009), suggesting that Elt might be a surrogate for rhTPO for stem cell mobilization. In this pilot trial, we evaluated the combination of Elt plus standard G-CSF and cyclophosphamide (C) for stem cell mobilization in patients (pts) with multiple myeloma (MM), a disease for which ASCT remains a standard of care (Blade et al. Blood 2010).Methods: Primary objectives included determination of the median number of CD34+ cells/kg mobilized and the maximum tolerated dose (MTD) of Elt. Pts had MM that was stable or responsive to at least two cycles of chemotherapy with plans for stem cell mobilization and ASCT. Four pts were to be enrolled in each of four dose escalation arms in which they received 0 (Arm D), 50 (Arm A), 100 (Arm B), or 150 mg (Arm C) of eltrombopag in combination with standard C + G-CSF. Adverse events (AEs) were graded by NCI-CTCAE v4.Results: 17 pts have been screened and enrolled to date. Two patients withdrew consent prior to receiving Elt and were excluded from statistical analysis. 15 patients have completed participation in the study to date and two patients remain to be enrolled in Arm C. The first subject in Arm A experienced delayed engraftment that was determined to be unrelated to ELT; rather, the event was attributed to administration of a one-time high dose of corticosteroid for management of a severe hypersensitivity reaction to DMSO that occurred during stem cell infusion. A second subject in Arm A had undergone mobilization with Elt prior to the previously described delayed engraftment event, and to ensure safety underwent a second mobilization with G-CSF and plerixafor. During ASCT, this patient received cells from the second mobilization procedure. While neither event met criteria for a dose-limiting toxicity, the protocol was amended such that three additional patients enrolled in Arm A underwent two rounds of mobilization - the first with Elt plus C and G-CSF and a second with G-CSF plus plerixafor - and received as part of ASCT cells mobilized with Elt. Each of these patients engrafted successfully. The median number of CD34+ cells/kg collected during the first collection day of apheresis in Arms D, A, B, and C was 8.0, 11.0, 15.3, and 26.4. The median total number of CD34+ stem cells collected following mobilization with Elt plus C and G-CSF in Arms D, A, B, and C was 13.2, 12.7, 15.4, and 26.4. The percentage of patients in Arms D, A, B, and C who achieved a target collection of 8 x 10^6 CD34+ stem cells in one collection day was 50, 60, 75, and 100%. There have been no severe adverse events related to Elt .Conclusions: Administration of Elt in combination with C plus G-CSF for stem cell mobilization in pts with MM undergoing ASCT was safe and well tolerated, with no DLTs or severe AEs attributable to Elt. The small size of this pilot study precludes formal statistical comparison of outcomes across treatment Arms, but there appears to be a trend toward increase in yield of CD34+ cells and decrease in apheresis procedures required with increasing doses of Elt. DisclosuresRichardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

A Phase I Study of the Safety and Feasibility of Bortezomib in Combination with G-CSF for Stem Cell Mobilization in Patients with Multiple Myeloma

Background: Granulocyte colony-stimulating factor (G-CSF) is the most frequently used agent for stem cell mobilization in multiple myeloma (MM). G-CSF results in suboptimal stem cell yields in 5-30% of MM patients and results in the mobilization of both progenitor cells and tumor cells. While the consequence of reinfusing malignant cells remains unclear, one prospective study has suggested that the presence of myeloma cells in PBSC grafts correlates with poor outcome after transplantation. Thus, in vivo or ex vivo purging of myeloma cells in PBSC products may represent a valid strategy for reducing relapse.In this phase I study, we explored the mobilization effect of bortezomib (BTZ) when given at peak G-CSF mobilization. We previously reported that in mice, administration of BTZ after 4 days of G-CSF augments mobilization with G-CSF significantly by modulating VLA-4/VCAM-1 axis (Ghobadi, et al, Blood, 2014). We hypothesize that administration of BTZ will augment mobilization and have an in vivo purging effect on myeloma cells.Objectives: 1) To determine the maximum tolerated dose (MTD) of BTZ when administered with G-CSF for stem cell mobilization; 2) To analyze the mobilization effect of BTZ, 3) To determine the effect of BTZ on frequency and number of circulating myeloma cells.Methods: This was a phase I study using 3 dose levels of BTZ (0.7, 1.0, and 1.3 mg/m2). G-CSF was administered (10 μg/kg) for five days. On the evening of the fourth day, a single dose of BTZ was administered. Peripheral blood was drawn 1-2 hours before and 15-18 hours following BTZ administration (prior to day 5 G-CSF administration) to analyze the effect of BTZ on peak CD34+ count and circulating myeloma cells. Standard apheresis was then performed (~20L). If the target collection was not met after 1 apheresis, up to 3 additional days of G-CSF and apheresis were performed. No additional BTZ was administered. Following mobilization patients underwent ASCT per institutional guidelines. Eligible patients had upfront MM and were candidates for ASCT. Prior BTZ exposure was not exclusionary. Dose limiting toxicity was defined as grade 4 hematologic toxicities and any grade 3-5 non hematologic toxicities within 28 days of BTZ administration.Results: Ten patients were enrolled; 1 patient at 0.7mg/m2, 3 at 1.0mg/m2, and 6 at 1.3mg/m2. The median age was 64 years; 60% were male. All patients had received prior BTZ, largely in combination regimens with lenalidomide or cyclophosphamide, the median number of prior cycles was 4.There were no dose limiting toxicities or serious adverse events reported, therefore an MTD was not established.Prior to G-CSF administration, the mean PB CD34+ was 1/μL (range: 0-3/ μL). Mean PB CD34+ cells at Day 4 prior to BTZ administration and 15 hours after BTZ administration (Day 5 prior to G-CSF administration and apheresis) were 25/μL (range: 1-59/ μL) and 38/μL (range: 2-84/ μL) respectively suggesting that administration of BTZ at peak G-CSF mobilization can potentially increase circulating CD34+ cells (Figure 1). The median CD34+ collection following 1 apheresis was 4.5 x 106/kg. Fifty percent of the patients collected > 5.0 x 106/kg CD341+ cells after 1 apheresis, 70% in 1-4 apheresis. Correlative studies using clonoSEQ technology are currently being performed to analyze the effect of BTZ on circulating myeloma cells.At the time of abstract preparation, 8 patients have subsequently undergone ASCT. The median times to neutrophil and platelet engraftment were 11.5 and 22.5 days, respectively. The complete response rate (CRR) was 29%. To date, there has been one case or relapse/progression at a median follow-up of 5.6 months.Discussion: Administration of BTZ at peak G-CSF mobilization was safe and well tolerated and enhanced G-CSF-induced stem cell mobilization. BTZ administration did not seem to impact graft viability as all patients had successful engraftment and the time to engraftment was unaffected. Correlative studies are currently being performed to determine if BTZ had an in vivo purging effect on myeloma cells as we hypothesized. [Display omitted] DisclosuresUy:Glycomimetics: Consultancy; Boehringer Ingelheim: Consultancy. Vij:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Karyopharma: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy; Shire: Consultancy; Celgene: Consultancy; Amgen: Consultancy, Research Funding. DiPersio:Incyte Corporation: Research Funding.

Increased Efficacy of Chemomobilization with Intermediate Dose Cytarabine + G-CSF Compared to G-CSF Alone for Patients with Multiple Myeloma: Results of a Prospective, Randomized Trial

INTRODUCTIONHigh-dose therapy with autologous stem cell support (autologous stem cell transplantation, autoSCT) remains the standard of care for eligible patients with multiple myeloma (MM). Planned tandem autoSCT which can also be considered for all transplant candidates requires collection of at least 5 x 10^6 CD34+ cells/kg. Randomized trials comparing chemomobilization with use of cyclophosphamide (CY) + G-CSF to G-CSF alone did not demonstrate clear advantage of addition of CY to growth factor. In recent years, intermediate-dose cytosine arabinoside (ID-AraC) + G-CSF has been proposed, showing high mobilization potential, as demonstrated in retrospective analysis (Giebel et al, Bone Marrow Transplant 2013, 48: 915-21). The goal of this prospective, randomized trial (ClinicalTrials.gov identifier: NCT01908621) was to compare the efficacy of ID-AraC + G-CSF with G-CSF alone in patients with MM referred for double autoSCT. METHODSThe inclusion criteria were set as follows1) the diagnosis of MM, 2) age 18-65 years, 3) at least partial remission achieved after one or more lines of therapy including six or more cycles containing components like thalidomide, lenalidomide , bortezomib, or melphalan, 4) planned tandem autoSCT procedure.The proportion of patients with stem cell yield of at least 5 × 10^6 CD34+ cells/kg was the primary study end-point.Mobilization regimens were as follows1) ID-AraC arm - AraC 2 x 0.4 g/m2 on days 1,2 (total 1.6 g/m2) + filgrastim 10 ug/kg/day starting from day 5; 2) G-CSF arm - filgrastim 10 μg/kg/day for five consecutive days. Leukaphereses were started when the level of circulating CD34+ cells in peripheral blood (PB) reached at least 10/uL and were continued for the maximum period of three days or until reaching the target CD34+ cell yield. Leukaphereses were performed using Spectra Optia Apheresis System, Mononuclear Cell Collection Procedure (Therumo BCT Inc., Lakewood, CO, USA), processing 2 total blood volumes. RESULTSBetween March 2013 and March 2016, 44 and 46 patients were randomly assigned to ID-AraC and G-CSF study arm, respectively. Patients in the ID-AraC arm were younger (median age 56 years, range (33-65) for ID-AraC and 60 years (37-65) for G-CSF, p=0.04). The groups did not differ in terms of MM remission status at mobilization (p=0.3), the number of preceding lines of chemotherapy (p=0.5) and the frequency of preceding radiotherapy (p=0.4).The level of CD34+ cells in PB required to start leukaphereses was achieved in all patients in the ID-AraC arm and in 44 (96%) of them in the G-CSF arm, p=0.2.In the ID-AraC group, 43 patients (98%) collected at least 5 x 10^6 CD34+ cells/kg compared to 32 patients (70%) in the G-CSF group (p=0.0003). In a multivariate model adjusted for age, the use of G-CSF alone was independently associated with increased risk of mobilization failure (Odds ratio = 17.5; 95% CI = 2.1-144.9; p=0.007).The median peak number of circulating CD34+ cells was 346 (11-1044)/µL vs. 40 (1-326)/µL (p<0.000001), while the median number of collected CD34+ cells was 20.2 (2.9-59.4) x10^6/kg vs. 5.9 (0-11) x10^6/kg, respectively (p<0.000001). A single apheresis was sufficient to achieve the threshold number of harvested CD34+ cells in 37 of cases (86%) after ID-AraC+G-CSF compared to 13 (41%) after G-CSF alone (p=0.00008). The median day of the first apheresis was 13 (range, 12-15; SD=0.7) after ID-AraC. CONCLUSIONSMobilization with ID-AraC + G-CSF is associated with significantly higher efficacy than G-CSF alone. In the studied group it allowed for collection of CD34+ cell number adequate for tandem autoSCT in almost all MM patients, usually with a single leukapheresis. This study provides the first evidence coming from prospective, randomized trial for the advantage of chemomobilization over G-CSF monotherapy in terms of proportion of patients achieving CD34+ yield sufficient for autoSCT. DisclosuresNo relevant conflicts of interest to declare.

Factors associated with bone marrow stem cell yield for pediatric allogeneic stem cell transplantation: The impact of donor characteristics.

The aim of this study was to investigate the effects of donor characteristics on CD34+ cell yield in BM harvest. Between April 2010 and November 2013, consecutive donors who underwent BM harvesting in our BM transplantation unit were retrospectively investigated. Donors were classified into two groups: those who donated BM without mobilization (steady-state BM donors) and those who received G-CSF for stem cell mobilization (G-CSF-primed BM donors). Donor characteristics (age, gender, race, body weight, BMI, and laboratory factors including donor's leukocyte, platelet, and monocyte) and their relationship with total nuclear cell and CD34+ cell numbers has been evaluated. A total of 64 healthy related donors (29 males/35 females, median age 11.2years; 49 [76.6%] younger than 18 and 36 [56.3%] younger than 12years) were included in the study. The median CD34+ cell yield in the harvest was 0.12×106 /L (0.02-0.21) in SS-BM donors and 0.18×106 /L (0.09-0.67) in GP-BM donors (P=.03). Median of CD34+ cell count given to recipients was 2.6×106 /recipient body weight (1.3-19.3) in SS-BM yields and 3.8×106 /recipient body weight (1.1-10.2) in GP-BM yields, respectively. Multiple regression analysis showed that donor height and pre-G-CSF platelet were the most important parameters to obtain a sufficient BM harvest. Our data suggest that the shorter donors and the donors with higher thrombocyte counts may offer more hematopoietic stem cell. The height and thrombocyte count of the donors should be taken into consideration before planning the targeted CD34+ cell count especially for pediatric donors.

Small Fractions of Muscular Dystrophy Embryonic Stem Cells Yield Severe Cardiac and Skeletal Muscle Defects in Adult Mouse Chimeras.

Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

Mesenchymal Stem Cells Yield Transient Improvements in Motor Function in an Infant Rhesus Macaque with Severe Early-Onset Krabbe Disease.

Krabbe disease, or globoid cell leukodystrophy, is a rare disorder caused by deficient galactosylceramidase activity and loss of myelin‐forming oligodendrocytes, resulting in progressive demyelination and severely impaired motor function. Disease symptoms in humans appear within 3–6 months of age (early infantile) and manifest as marked irritability, spasticity, and seizures. The disease is often fatal by the second year of life, with few effective treatment options. Herein we evaluated the therapeutic potential of mesenchymal stem cells (MSCs) administered intracranially to a 1‐month‐old rhesus macaque diagnosed with severe early‐onset Krabbe disease that displayed neurologic and behavioral symptoms similar to those of human patients. The infant was subjected to physical and neurological behavior examinations and nerve conduction velocity tests to assess efficacy, and outcomes were compared with age‐matched normal infants and Krabbe‐affected rhesus monkeys with late‐onset disease. Changes in major blood lymphocyte populations were also monitored to assess host immune cell responses. MSC administration resulted in transient improvements in coordination, ambulation, cognition, and large motor skills, which correlated with increased peripheral nerve conduction velocities and decreased latencies. Improvements also corresponded to transient increases in peripheral blood lymphocyte counts, but secondary challenge failed to elicit allo‐antibody production. Nevertheless, white cell and neutrophil counts showed dramatic increases, and CD20+ B cell counts underwent a precipitous decline at late stages of disease progression. Correlative data linking MSC administration to transient improvements in motor function suggest that MSCs should be evaluated further as an experimental therapy for rare neurodegenerative diseases. Stem Cells Translational Medicine 2017;6:99–109

Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue

We compared cell number, putative stem cell markers, and clonogenic ability in fresh uncultured human limbal epithelial cells to that obtained from stored organ-cultured tissue. Cell suspensions were formed from fresh and organ culture-stored human limbal epithelium. Expression of putative stem cell markers ΔNp63 and TrkA was performed using immunofluorescent staining before culture. Colony-forming efficiency (CFE) assays were performed at first passage. The effects of tissue storage, age, and postmortem/culture times were analyzed in a general linear model. Limbal tissue from 94 donors (34 fresh and 60 stored) was compared. Three times more cells were obtained per eye from fresh (35.34 × 104; SD, 17.39) than stored (11.24 × 104; SD, 11.57; P < 0.01) tissue. A higher proportion of cells from fresh tissue were viable (91.9%; SD, 5.7 vs. 85%; SD, 10.8) P < 0.01. Higher total cell expression of ΔNp63 (20.19 × 104; SD, 15.5 vs. 3.28 104; SD, 4.33) and TrkA (59.24 × 104; SD, 13.21 vs. 7.65 × 104; SD, 1.05) was observed in fresh than stored tissue (P < 0.01). Colony-forming efficiency was higher for fresh (1.42; SD, 0.12) than stored (0.43; SD, 0.15; P < 0.01) cells. For stored tissue only, there was a significant inverse relationship between donor age and total number of cells isolated (R2 = 0.27, P < 0.001). Storage of corneoscleral discs in organ culture medium leads to significant reduction in limbal epithelial cell number, expression of ΔNp63 and TrkA, and viability compared to fresh tissue. There is a smaller basal stem cell population in stored compared to fresh tissue.

Factors influencing yield and neuronal differentiation of mesenchymal stem cells from umbilical cord blood and matrix.

Umbilical cord blood and Wharton's jelly (WJ) are potential sources of mesenchymal stem cells (MSCs). We investigated whether harvesting and donor characteristics affected yield and neuronal differentiation, and compared human umbilical cord blood (hUCB) and WJ-derived MSCs regarding neuronal differentiation and cytokine secretion. MSCs were analyzed by immunoblotting after seven days of differentiation; cytokine protein arrays were used to analyze conditioned medium. Birth weight and blood/anticoagulant ratio influenced MSC yield per unit blood volume, but not maternal and gestational age, delivery mode or fetal gender. Expression of the early neuronal differentiation marker nestin was unaffected by these variables. hUCB- and WJ-derived MSC secrete distinct cytokine profiles. Cell yield is affected by certain donor characteristics. hUCB- and WJ-derived MSCs may serve distinct therapeutic niches.

Signaling from maize organ primordia via FASCIATED EAR3 regulates stem cell proliferation and yield traits

Shoot apical meristems are stem cell niches that balance proliferation with the incorporation of daughter cells into organ primordia. This balance is maintained by CLAVATA-WUSCHEL feedback signaling between the stem cells at the tip of the meristem and the underlying organizing center. Signals that provide feedback from organ primordia to control the stem cell niche in plants have also been hypothesized, but their identities are unknown. Here we report FASCIATED EAR3 (FEA3), a leucine-rich-repeat receptor that functions in stem cell control and responds to a CLAVATA3/ESR-related (CLE) peptide expressed in organ primordia. We modeled our results to propose a regulatory system that transmits signals from differentiating cells in organ primordia back to the stem cell niche and that appears to function broadly in the plant kingdom. Furthermore, we demonstrate an application of this new signaling feedback, by showing that weak alleles of fea3 enhance hybrid maize yield traits.

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.

Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors.

Production of human mesenchymal stem cells for allogeneic cell therapies requires scalable, cost‐effective manufacturing processes. Microcarriers enable the culture of anchorage‐dependent cells in stirred‐tank bioreactors. However, no robust, transferable methodology for microcarrier selection exists, with studies providing little or no reason explaining why a microcarrier was employed. We systematically evaluated 13 microcarriers for human bone marrow‐derived MSC (hBM‐MSCs) expansion from three donors to establish a reproducible and transferable methodology for microcarrier selection. Monolayer studies demonstrated input cell line variability with respect to growth kinetics and metabolite flux. HBM‐MSC1 underwent more cumulative population doublings over three passages in comparison to hBM‐MSC2 and hBM‐MSC3. In 100 mL spinner flasks, agitated conditions were significantly better than static conditions, irrespective of donor, and relative microcarrier performance was identical where the same microcarriers outperformed others with respect to growth kinetics and metabolite flux. Relative growth kinetics between donor cells on the microcarriers were the same as the monolayer study. Plastic microcarriers were selected as the optimal microcarrier for hBM‐MSC expansion. HBM‐MSCs were successfully harvested and characterised, demonstrating hBM‐MSC immunophenotype and differentiation capacity. This approach provides a systematic method for microcarrier selection, and the findings identify potentially significant bioprocessing implications for microcarrier‐based allogeneic cell therapy manufacture.

Does bendamustine impact the mobilization of peripheral blood stem cells? A multicenter retrospective study of 23 cases

Bendamustine is used in the treatment of different relapsing or refractory subtypes of lymphoma. Its impact on the yield of peripheral blood stem cells is not well known. Twenty three patients who received bendamustine followed immediately or after another chemotherapy by stem cell mobilization (SCM) were included. The patients were divided into two groups: group 1 (n=17), in whom SCM was performed immediately after bendamustine chemotherapy, and group 2 (n=6), in whom SCM was performed after another cycle of chemotherapy. The success rate of mobilization after Bendamustine+/−plerixafor was 36% (eight cytapheresis succeeded for a total number of 22 cytapheresis); and 75% after other approaches (chemotherapy based or steady state) used for patients who received bendamustine previously. Although bendamustine used alone was not an effective drug to mobilize stem cells, this agent does not seem to have detrimental effects on subsequent SCM.

Hematopoietic stem cell transplantation for multiple sclerosis: is it a clinical reality?

Hematopoietic stem cell transplantation (HSCT) is a treatment paradigm that has long been utilized for cancers of the blood and bone marrow but has gained some traction as a treatment paradigm for multiple sclerosis (MS). Success in the treatment of patients with this approach has been reported primarily when strict inclusion criteria are imposed that have eventuated a more precise understanding of MS pathophysiology, thereby governing trial design. Moreover, enhancing the yield and purity of hematopoietic stem cells during isolation along with the utility of appropriate conditioning agents has provided a clearer foundation for clinical translation studies. To support this approach, preclinical data derived from animal models of MS, experimental autoimmune encephalomyelitis, have provided clear identification of multipotent stem cells that can reconstitute the immune system to override the autoimmune attack of the central nervous system. In this review, we will discuss the rationale of HSCT to treat MS by providing the benefits and complications of the clinically relevant protocols, the varying graft types, and conditioning regimens. However, we emphasize that future trials based on HSCT should be focused on specific therapeutic strategies to target and limit ongoing neurodegeneration and demyelination in progressive MS, in the hope that such treatment may serve a greater catchment of patient cohorts with potentially enhanced efficiency and lower toxicity. Despite these future ambitions, a proposed international multicenter, randomized clinical trial of HSCT should be governed by the best standard care of treatment, whereby MS patients are selected upon strict clinical course criteria and long-term follow-up studies of patients from international registries are imposed to advocate HSCT as a therapeutic option in the management of MS.

Does the preference of peripheral versus central venous access in peripheral blood stem cell collection/yield change stem cell kinetics in autologous stem cell transplantation?

Central venous access is often used during apheresis procedure in stem cell collection. The aim of the present study was to evaluate whether central or peripheral venous access has an effect on stem cell yield and the kinetics of the procedure and the product in patients undergoing ASCT after high dose therapy. A total of 327 patients were retrospectively reviewed. The use of peripheral venous access for stem cell yield was significantly more frequent in males compared to females (p = 0.005). Total volume of the product was significantly lower in central venous access group (p = 0.046). As being a less invasive procedure, peripheral venous access can be used for stem cell yield in eligible selected patients.

Time Dependent Decrease in Efficacy of Second G-CSF Mobilization in Healthy Unrelated Donors

AbstractIntroduction: Graft failure or a second allogeneic hematopoietic stem cell transplantation or HLA haplotypes leading to donor requests for two patients are reasons for a second collection of peripheral stem cells of the same donor. Few reports about second stimulation showed conflicting results. Higher G-CSF doses or the addition of plerixafor has been described either in autologous or allogenic settings. In order to get more information about second stimulation with G-CSF, we pooled the data from three collection centers in Germany.Donors and Methods: The collection centers of Dresden, Hameln and Cologne performed between December 1991 and May 2015 18,124 collections of peripheral stem cells. We identified 351 of donors who donated twice. All collections were performed after G-CSF stimulation (Lenograstim) of 7.5 to 10 µg / kg body weight. The same apheresis machine was used in 80 % of donors. Besides Spectra and Optia from Terumo ComTec machines from Fresenius were used. We compared leukocytes, platelets, hemoglobin and CD 34 positive cells in the blood before apheresis, yield of stem cells, and blood volume processed as endpoint of interest. There are differences in the number of donor results since the reported data in the first years were incomplete. We created 4 groups of donors depending on the interval between the two donations (Group A: interval <= 90 days, group B 91 to 180 days, group C 181 to 360 days, group D > 360 days). The minimum difference between two apheresis was 20 days, the maximum 4,436 days. The data of the donors were analyzed using NCSS as statistical program. To determine significance in the data paired t tests were performed.Results: Platelet counts, hemoglobin and erythrocyte counts at the time of apheresis was similar at first and second time point of collection. CD34-positive cells in the blood on the 1st day of apheresis were significantly higher with 77.9/µl at 1st apheresis compared to 67.9/µl at 2nd apheresis. Leukocyte counts were also higher at first donation date (42,954/µl compared to 40,330/µl). Considering the product, total collected CD34 pos. cells were lower at 2nd apheresis (617 * 106 compared to 566 * 106) but there was no significant difference in the CD34 pos. cells per kg BW of the patient which might be due to the observation that the body weight of the patients were lower at 2nd transplantation. We were interested in the time-dependence of the second mobilization capacity. The details are shown in the table. Leukocyte counts in the blood remained lower after 2nd G-CSF stimulation even after more than one year. In contrast, CD34 stimulation returned to values measured at the first stem cell collection. To achieve the requested amount of CD 34 pos. cells a higher blood volume had to be processed if the two collections are less than 6 months apart.Discussion: Our data are in accordance with earlier observations showing that at a second stimulation with G-CSF is less affective. The data of 351 donors indicate that this difference lasts for up to 1 year for stimulation of CD34 pos. cells. Only for leukocytes, there is still a significant difference also if restimulated after more than 1 year . In contrast, this decreased restimulation of stem cells has no important clinical effect on the possibility to get suffient numbers of stem cells for a transplantation. A second treatment of a donor at least with 7.5 to 10 µg G-CSF/kg body weight does not harm the donor since baseline hematologic parameters were the same at time of medical assessment.Table 1CD 34 pos. cells/µl (blood)Leukocytes/µl (blood)Total CD 34 pos. cell in the product (*10^6)CD 34 pos. cells / BW patient (*10^6)Processed blood volume of the donor (l)Interval between 2 collections < =90 days1st apheresis77.0* (69)42,772* (69)598* (71)8.5 (70)13.8* (55)2nd apheresis59.2*38,612*558*8.0616.1*Interval between 2 collections 91 - 180 days1st apheresis76.6* (94)42,872* (95)567* (95)7.27 (95)14.4* (80)2nd apheresis64.7*40,886*517*7.2215.6*Interval between 2 collections 181 - 360 days1st apheresis81.3* (85)43,643* (86)654* (87)9.33 (86)14.6 (79)2nd apheresis77.6*41,388*572*8.6814.9Interval between 2 collections > 360 days1st apheresis76.8 (98)42,557* (98)646 (98)9.66 (98)15.5 (89)2nd apheresis73.240,071*61510.3314.9*Difference significant (p<0.01) in paired t-test, in brackets number of donors DisclosuresSchetelig:GSK and Sanofi: Research Funding; Janssen, Sanofi and Neovii: Membership on an entity's Board of Directors or advisory committees. Ehninger:Cellex GmbH: Equity Ownership.

A Retrospective Single Center Comparison of Cyclophosphamide Plus G-CSF Versus G-CSF Alone for Peripheral Blood Stem Cell (PBSC) Mobilisation Following First Line Therapy in Patients with Multiple Myeloma

▪IntroductionAutologous transplantation is considered standard therapy for young and fit myeloma patients after initial therapy. Cyclophosphamide+ G-CSF is considered standard therapy for collection even though there is evidence for minimal anti-myeloma effect of cyclophosphamide, some increased short term toxicity and potential concerns regarding long term toxicity. There have been a few retrospective comparisons and one randomized study comparing cyclophosphamide based and G-CSF alone based PBSC collection. To our best knowledge they have not reported any impact in progression free survival (PFS) or overall survival (OS). We have compared here our myeloma patient cohort to explore these important endpoints.Patients and methods89 patients (55 male and 34 female) who underwent first autologous transplant for myeloma between 2003 and 2010 were analysed in our study. Mobilization was with G-CSF alone in 45 patients (median age 58 yrs, range 38-70 yrs.) and cyclophosphamide and G-CSF in 44 (median age 58 yrs, range 41-74 yrs.). Cyclophosphamide was used at 3g/m2 and in both cases G-CSF used was lenogastrim at 10mcg/kg. There were 7 patients with ISS stage 3 in the G-CSF only group as compared to 10 in the Cyclophosphamide group. Prior chemotherapy was cyclophosphamide, thalidomide and dexamethasone in majority of cases (n=55) with no difference across both groups. Data regarding high risk genetics and pre-transplant response was unavailable. We collected data progression free survival, overall survival, harvest dose and engraftment kinetics. Data was analyzed using SPSS and log rank test.ResultsThe median dose of stem cells collected with G-CSF alone was 3.59×106 CD34 cells/kg (range 1.84-8.09) where as with cyclophosphamide and G-CSF it was 3.8×106 CD34 cells/kg (range 1.6-13). There was no difference in engraftment between the two groups with median neutrophil engraftment (Absolute neutrophil count>0.20×109/L) at day 14 and platelet engraftment (>50×109/L) at day 15 and 16 respectively. Progression free survival was significantly better in G-CSF alone cohort (46 months vs. 38 months, P=0.016) (fig. a) Overall survival was better in the GCSF only group as well (113 months vs. 75 months, P=0.029) (fig b). In the 17 high risk patients PFS was much better in the G-CSF group (60months vs. 22 months, P=0.02) (Fig c). There were 4 (9%) admissions in the cyclophosphamide group due to neutropenic sepsis as compared to none in the G-CSF group.DiscussionCyclophosphamide and G-CSF may be associated with slightly higher stem cell yields but this margin is becoming smaller and not significant in the era of liberal plerixafor usage. In addition some patients are hospitalized due to neutropenic sepsis with this regimen. Our data shows anti-myeloma effects of cyclophosphamide +G-CSF is not demonstrated. There are ongoing studies from the Finland group which show no difference in the number of CD34+ cells collected after initial therapy with lenalidomide. The only difference is the number of days required for apheresis. In addition to above our single center experience shows both PFS and OS benefit for G-CSF only PBSC mobilization. . This may partially be explained by the slight difference in ISS risk stages in our patients but on censoring for ISS stage 3 these results were more pronounced. This is the first time we have seen any report point out towards a PFS and OS difference between two widely used mobilization regimens. This needs testing in a large randomized multi-center study to see if there is a difference and if so is this due to a change in milieu of lymphocytes. We have previously reported that absolute lymphocyte count on day 15 post autograft was reflective of a higher lymphocyte count in the apheresis bag in case of G-CSF only mobilizations as compared to cyclophosphamide +G-CSF. The absolute lymphocyte count on day 30 was a predictor for better OS. [Display omitted] [Display omitted] [Display omitted] DisclosuresSutton:bayer: Honoraria. Paneesha:Janssen: Consultancy. Nikolousis:Alexion: Honoraria. Kishore:Jazz pharma: Honoraria; Celgene: Honoraria.

Commentary on: isolation and differentiation potential of human mesenchymal stem cells from adipose tissue harvested by water jet-assisted liposuction.

In this article, Meyer et al examine the yield of mesenchymal stem cells (MCSs) with water jet-assisted liposuction (WAL).1 WAL uses simultaneous infiltration and suction to dissect and extract fat during liposuction. Using water to dissect tissues is an idea that evolved in the last century and was later geared towards the dissection of fat in 2001.2 Since that time, a number of studies have suggested advantages to the use of WAL, including decreased pain, decreased intraoperative swelling, better contouring, and decreased need for anesthesia.3-5 The gentle nature of WAL has been utilized in the treatment of lipoedema, where clinicians noted that this aspiration method may induce less trauma to lymphatics.6 With the evolution of fat grafting, the utility of WAL for fat harvest has been explored.5 Harvest method can certainly impact graft …

Cyclophosphamide plus granulocyte-colony stimulating factor for hematopoietic stem cell mobilization in patients with multiple myeloma.

We retrospectively reviewed the results of cyclophosphamide (3 g/m(2) ), doxorubicin and dexamethasone plus granulocyte-colony stimulating factor (G-CSF) (ID-CY/DOX group), low-dose cyclophosphamide (2 g/m(2) ) plus G-CSF (LD-CY group) and G-CSF alone (G-CSF group) for stem cell mobilization in patients with multiple myeloma. A total of 89 patients with 93 mobilizations were included. Apheresis was started when total white blood cell (WBC) count >10 × 10(9) /L for ID-CY/DOX and LD-CY groups and after eight doses of G-CSF (5 μg/kg twice daily) for G-CSF group. For five mobilizations in ID-CY/DOX group, the rate of successful mobilization (≥4.0 × 10(6) /kg CD34+ cells) was 80%. For 78 mobilizations in LD-CY group, the successful rate was 80.8%. For 10 mobilizations in the G-CSF group, the successful rate was 50%. The mean yield of CD34+ cells was higher in ID-CY/DOX and LD-CY groups as compared with that in G-CSF group (P = 0.026 and 0.020, respectively). There was no difference in the yield of CD34+ cells between ID-CY/DOX and LD-CY groups (P = 0.831). After autologous stem cell transplantation, the days to neutrophil and platelet engraftment were similar in these three groups (P = 0.713 and 0.821, respectively). In conclusion, we observed that ID-CY/DOX and LD-CY plus G-CSF for stem cell mobilization resulted in a higher successful rate and higher stem cell yields than G-CSF alone and their engraftment time were similar. Total WBC count >10 × 10(9) /L can be used as a guide to start apheresis in CY-based stem cell mobilization. J. Clin. Apheresis 31:423-428, 2016. © 2015 Wiley Periodicals, Inc.