Number Of Total Nucleated Cells Research Articles

Donor-matched comparison of chondrogenic progenitors resident in human infrapatellar fat pad, synovium and periosteum – Implications for cartilage repair

Purpose: Over the past decades, multiple tissue sources have been used as a source of cells in clinical trials for cartilage repair, including infrapatellar fat pad, synovium, bone marrow, peripheral blood, adipose-stromal vascular tissue, and periosteum. Despite the increased clinical use of bone marrow/peripheral blood cells and adipose tissue obtained subcutaneously or through liposuction, there are reports suggesting higher concentration and chondrogenic potential of progenitors derived from synovium and infrapatellar fat pad. Such tissues around the knee are easily accessible and can be harvested via arthroscopy or open surgery using minimally invasive techniques, with minimal complications at the donor site. However, to date, there is no consensus on the optimal cell source for cartilage repair. Furthermore, no in-vitro or in-vivo study has systematically compared tissue sources used in clinical practice. Therefore, the aims of this study were to quantitatively define and compare the progenitor populations in infrapatellar fat pad, synovium and periosteum in donor-matched osteoarthritic (OA) knee joints with respect to 1) cell concentration, 2) chondrogenic connective tissue progenitor (CTP-Cs) prevalence and concentration, and 3) biological potential of the CTP-Cs progenitors, and 4) to determine the effect of demographic factors (age and gender) on these variables. Methods: Twenty-eight patients with varus deformity undergoing total knee arthroplasty (TKA) were recruited (mean age:63.1years, range:37-82years, male=12, female=16) under an IRB approved protocol. During TKA, 0.5-1.5cc of synovial membrane was harvested from the medial suprapatellar area, assuring that fat tissue was not present. 2.5cc of fat was harvested from the infrapatellar fat pad after removal of overlying synovium. 0.5-1.5cc of periosteum was harvested from the anterior femoral region, proximal to the trochlear grove. Wet weight of tissues was recorded and cells were isolated by enzymatic digestion. The total number of nucleated cells per mg of tissue was counted using hemocytometer (cell concentration, [Cells]). This cell pellet was then suspended in chondrogenic medium and plated at a density of 100,000/well (24,000cells/cm2) at 37oC, 98% humidity, 20% O2, and 5% CO2 for 6 days. On day 6, cultures were harvested, fixed and stained for nuclei with bis-benzimide (BB), and for glycosaminoglycan-containing extracellular matrix (GAG-ECM) with acridine orange (AO). Automated quantitative colony forming unit (CFU) analysis was performed using ColonyzeTM image analysis software to assess CTP-derived colonies from fat, synovium, and periosteum (Fig 1). A significance level of 0.05 was used overall, with Holm’s method used to control the family-wise error rate. Pearson’s correlation coefficient (r) was used to estimate the within-patient association between two colony metrics for each individual patient, while the repeated measures correlation coefficient (rrm) was used to estimate the within-patient association between two colony metrics that was common across all patients. All analyses were performed in R 3.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Results: Cell concentration (nucleated cells/mg) did not differ between fat, synovium, and periosteum across all age groups and gender. Patients <60years did not differ on CTP prevalence (PCTP-C)(CTP-Cs/million nucleated cells) between the three sources. However, in patients >60years, both fat and synovium had a higher PCTP-C than periosteum (p<0.001). For females, PCTP-C was higher in fat (p<0.001) and synovium (p=0.001) than periosteum (after adjusting for age). No differences in PCTP-C were observed in males. Overall CTP-C concentration (CTP-C/mg) did not differ significantly between the three sources (p=0.226), however significant differences were encountered with age (p<0.001). Proliferation potential of CTP-Cs differed significantly between tissue sources (synovium>fat>periosteum), specifically in patients >60years. Cell migration (density) and differentiation potential were comparable (p>0.5) (Table1). Conclusions: This study isolated cells using an established protocol from three potential tissue sources resident in the knee, and cultured them in the same chondrogenic conditions for systematically quantifying and comparing the performance of the progenitors in vitro. To the best of our knowledge, this study is the first to systematically assess these tissue sources by implementing reproducible methods for automated colony-forming unit assay in accordance with ASTM Standard Method F2944-12 to quantitatively compare and characterize the cell and CTP-C population resident in these donor-matched tissues. These results provide insight into potential patient population that may benefit from one or the other tissue as source of progenitors for cartilage repair. Chondrogenic progenitors are available in fat, synovium, and periosteum, with differences encountered based on age and gender. The heterogeneity among the CTP-Cs suggests performance-based selection might be useful to optimize cell-sourcing strategies to improve efficacy of cellular therapies for cartilage repair.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Two-fold Increase in the Number of Total Nucleated Cells in the Bone Marrow Concentrate Obtained From the Bone Marrow Aspirate May Not Be Ideal: Response.

Two-fold Increase in the Number of Total Nucleated Cells in the Bone Marrow Concentrate Obtained From the Bone Marrow Aspirate May Not Be Ideal: Response.

Two-fold Increase in the Number of Total Nucleated Cells in the Bone Marrow Concentrate Obtained From Bone Marrow Aspirate May Not Be Ideal: Letter to the Editor.

Two-fold Increase in the Number of Total Nucleated Cells in the Bone Marrow Concentrate Obtained From Bone Marrow Aspirate May Not Be Ideal: Letter to the Editor.

Umbilical cord blood quality and quantity: Collection up to transplantation.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells for transplantation in some blood disorders. One of the major factors that influence on transplantation fate is cord blood (CB) cell count, in addition to human leukocyte antigen similarity and CD34+ cell number. Here, we review the factors that could effect on quality and quantity of CBUs. Relevant English-language literatures were searched and retrieved from PubMed using the terms: CB, quality, collection, and transplantation. The numbers of total nucleated cells (TNCs) and CD34+ cells are good indicators of CB quality because they have been associated with engraftment; thereby, whatever the TNCs in a CB unit (CBU) are higher, more likely they led to successful engraftment. Many factors influence the quantity and quality of UCB units that collect after delivery. Some parameters are not in our hands, such as maternal and infant factors, and hence, we cannot change these. However, some other factors are in our authority, such as mode of collection, type and amount of anticoagulant, and time and temperature during collection to postthaw CBUs and freeze-and-thaw procedures. By optimizing the CB collection, we can improve the quantity and quality of UCB for storage and increase the likelihood of its use for transplantation.

Cord Blood Units Containing Lower CD34+ Cells (0.5 - 1.0 x 105 /kg) Could be Alternative Donor Candidates for Single-Unit Cord Blood Transplantation for Adults: A Retrospective Study of 421 Patients in a Single Institute

BACKGROUND: Cord blood is an established alternative donor cell source for allogeneic hematopoietic cell transplantation. However, engraftment failure is still a major concern after transplantation, especially for patients transplanted lower doses of donor cells. Higher CD34+ cell dose leads to a secure and fast engraftment, and the units of cord blood which contain CD34+ cells of 1.0 - 1.7 x 105 /kg at freezing or 1.0 - 1.2 x 105 /kg at thawing are recommended (Thomas' Hematopoietic Cell Transplantation, 5th Edition). Actually, most adult patients cannot obtain such sufficient cell dose-containing cord bloods and the feasibility of single-unit cord blood transplantation (CBT) containing lower CD34+ cell dose than 1.0 x 105 /kg is unclear.METHODS: To investigate the lower threshold of CD34+ cell dose, we studied the patients who received single-unit CBT as the first transplantation between 2009 and 2017. The patients whose ECOG performance status was 0 or 1, and who do not have donor-specific anti-HLA antibody (DSA) were analyzed. Institutional review board of Toranomon Hospital approved the study (research number #1666).RESULTS: A total of 421 patients were studied. The median age and body weight of patients was 57 years (range, 16 - 74) and 56.4 kg (32.2 - 94.6), respectively. Myeloid diseases accounted for 78% of the patients, and 83% were not in remission. Myeloablative conditioning regimens were used in 80% of the patients. All patients used Tac (26%) or Tac plus MMF (74%) as GVHD prevention. The median numbers of total nucleated cells and CD34+ cells were 2.61 x107 /kg (range, 1.57 - 5.85) and 0.86 x 105 /kg (0.29 - 3.77) at freezing, respectively. The cumulative incidence of neutrophil engraftment was 90.7% at 60 days after transplantation (95% confidence interval, 87.5 - 93.1). The median day of neutrophil engraftment was day 21 (range, 5 - 45). Multivariate analysis identified higher CD34+ cell dose, less HLA mismatch, and lymphoid disease as significant favorable factors for neutrophil engraftment (p < 0.05), and CD34+ cell dose was most significant among the following pre-transplant factors (HR 1.57, p < 0.00001): age (≤ 57 vs. >57 years), body weight (≤ 56.4 vs. > 56.4 kg), ECOG performance status (0 vs. 1), disease (myeloid vs. lymphoid), disease status (in CR vs. not in CR), anti-HLA antibody (not DSA) (positive vs. negative), total nucleated cell dose (≤ 2.61 vs. > 2.61 x 107 /kg), CD34+ cell dose (≤ 0.86 vs. >0.86 x 105 /kg), HLA antigen match (≤4/6 vs. ≥5/6), ABO match (match vs. mismatch), sex match (match vs. mismatch), GVHD prevention (Tac vs. TAC plus MMF), and the intensity of conditioning regimen (MAC vs. RIC). Then, we compared the cumulative incidence of neutrophil engraftment between 4 groups as follows: 90.2% for group A (> 1.5 x 105 /kg, n = 41); 91.7% for group B (1.0 - 1.5 x 105 /kg, n = 109); 91.4% for group C (0.5 - 1.0 x 105 /kg, n = 255); 75.0% for group D (< 0.5 x 105 /kg, n = 16) (p < 0.01). The median day of neutrophil engraftment was faster for the patients transplanted more CD34+ cell doses: day 17 for group A; day 19 for B; day 21 for C; day 26.5 for D (p < 0.0001). Next, we focused on group C transplanted lower CD34+ cell dose than the recommendation (0.5 - 1.0 x 105 /kg, n = 255).The patients were divided into 5 groups according to their CD34+ cell doses, and we compared their cumulative incidence of neutrophil engraftment as follows: 96.0% for group C1 (0.9 - 1.0 x 105 /kg, n = 50); 89.7% for group C2 (0.8 - 0.9 x 105 /kg, n = 39); 88.1% for group C3 (0.7 - 0.8 x 105 /kg, n = 67); 92.2% for group C4 (0.6 - 0.7 x 105 /kg, n = 51); 91.7% for group C5 (0.5 - 0.6 x 105 /kg, n = 48) (p = 0.03). The median day of neutrophil engraftment was significantly faster for the patients transplanted more CD34+ cell dose: day 20 for group C1; day 21 for C2; day 21 for C3; day 23 for C4; day 24 for C5 (p < 0.01). Overall survival was not significantly different between group A vs. B vs. C vs. D, nor group C1 vs. C2 vs. C3 vs. C4 vs. C5, respectively.DISCUSSION & CONCLUSION: Significantly faster neutrophil engraftment was demonstrated for patients transplanted more CD34+ cells after single-unit CBT. On the other hand, the cumulative incidences of neutrophil engraftment at day 60 were comparable among the patients who used > 0.5 x 105 /kg of CD34+ cells to be around 90%. The cord blood units containing 0.5 - 1.0 x 105 /kg at freezing could be alternative donor candidates for cord blood selection, if delayed engraftment was clinically acceptable for recipients. DisclosuresYamamoto:Bristol-Myers Squibb: Honoraria.

Infusion of a Cryopreservable Human Megakaryocyte-Biased Cell Product Results in Sustained Platelet Reconstitution In Vivo

The demand for platelet (PTL) transfusions has steadily increased, straining a supply that is limited by its dependency on donors, short lifespan, risk of infections and alloimmunization. This stimulated the search for alternative PTL sources including PTLs generated ex vivo from primary CD34+ cells and immortalized pluripotent stem cells. These approaches, however, are associated with obstacles such as scalability and encounter identical limitations as donor PTLs: short shelf life, storage at ambient temperature and limited lifespan after infusion. These obstacles lead us to focus our efforts on not producing PLTs but rather a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTLs after transfusion into patients.Umbilical cord blood units (CBU) are readily available sources for stem cell for transplantation. We created an efficient and cost effective culture system in which CB-derived CD34+ cells are first expanded then allowed to mature into MKs. Initially, we determined the optimal culture period (10-11 days) resulting in the greatest number of CD41+/CD42b- and CD41+/CD42b+ MKs which are capable of PTL production. Next, we used research and clinical grade CBU to generate clinically relevant doses of MK. The median number of CD34+ cells selected from one CBU was 2.5x106 with a purity of 90% (n=4). Following expansion and MK maturation, these cells generated 5.8x107 viable total nucleated cells (TNC)/CBU. Out of these, 3.3 x 107 were CD41+ MKs which corresponds to a median cell dose of 4.1x105 CD41+ cells/Kg of body weight. 92% of CD41+ MKs were mature CD42b+ cells which we previously showed that are capable of ex vivo platelet production. Finally, we performed clonogenic assays and found that one CBU can generate ~1.5x106 CFU-MK.One half of these MK-biased cultures was characterized and assessed immediately after culture and the other half was cryopreserved. The fresh product was infused into sublethally irradiated NSG mice and the presence of human PTLs in the mouse peripheral blood (mPB) was evaluated weekly for 8 weeks at which time the animals were also analyzed for hMK chimerism in the bone marrow (BM), spleen (SP) and lung. The results demonstrate that 87% of animals (13 out of 15) had a robust hPTLs population in their PB. hPTL were detected as early as week 1 post infusion and their number peaked on week 4 (median, 6x103 hPTL/μl) after which it plateaued. The release of hPTL in the mPB was accompanied by the presence of hCD41+ MKs in the mBM, SP and the lung indicating that the infused cells provided both early hPTL release and a reservoir of MK precursors for continuous hPTL production. We also found that in addition to MKs, these same organs contained hCD34+, CD45+ and myeloid CD15+ cells. These findings underscore the capabilities of this product which might have broader clinical applicability such as utilization during myeloablative or suppressive chemo/radiotherapy to improve the time and duration for both neutrophil and platelet engraftment. Equally important, we provide novel evidence that the lung is a site for hMK engraftment after transplantation, in line with recent reports recognizing the pulmonary bed as site for platelet production in the mouse.The major advantage of developing a MK-based product over ex vivo generated PTLs is the amenability of the former to cryopreservation thus becoming a readily available cellular therapy which would be amenable to stock-piling. Therefore, portions of the same MK products described above were cryopreserved then subjected to ex vivo and in vivo studies identical to these performed on their fresh counterparts. Following thawing, the average recovery rate was 71% for TNC and 74.3% for CD41+ cells. MK phenotype and morphology as well as the number CFU-MK generated ex vivo were identical to that found in the fresh product. Although the number of TNC in the thawed product was lower than that of its fresh counterpart, the number of hPTL detected after its infusion ranged from 0.4 to 20.5x103 hPTL/μl which is comparable to that detected after infusion of the fresh equivalent, 0.7-16x103 hPTL/μl.In summary, we created a potent transfusable MK cell product that provides robust and sustained PTL and hematopoietic engraftment in vivo and maintains this capability after cryopreservation. Clinical development of such product is now being pursued for the treatment of thrombocytopenia in acute leukemia patients undergoing chemotherapy. DisclosuresHoffman:Summer Road: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding; Merus: Research Funding; Incyte: Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding.

Early Engraftment of Unrelated Cord Blood Transplantation in Patients with Acquired Severe Aplastic Anemia Using Conditioning Regimen without Anti-Thymocyte Globulin

Background:Previous studies show that the use of unrelated cord blood transplantation (UCBT) for severe aplastic anemia (SAA) has poor outcome because of high incidence of primary graft failure. Effective measures to completely prevent rejection in SAA remain to be identified, but higher cell dose, less HLA disparities and better conditioning regimen are known to improve the outcome. In this study we compare two conditioning regimens to determine which is better to facilitate early engraftment after UCBT.Patients and methods:We retrospectively analyzed the outcomes of 35 Chinese patients with acquired SAA who received UCBT since September 2016. Eighteen patients (ATG group) used a conditioning regimen consisting of ATG (thymoglobulin) 2.5 mg/kg (D-9 to D-7) with fludarabine 30 mg/m2 (D-9 to D-4), cyclophosphamide 60 mg/kg (D-3 to D-2) and total body irradiation (3 Gray) on D-1. Median age at time of UCBT was 9 (4-37) years. The median total nucleated cell number and CD34-positive cell number at infusion were 4.08 (1.74-9.36) × 107/kg and 2.13 (0.67-4.29) × 105/kg, respectively. Another group (No-ATG) of 17 patients used a conditioning regimen without ATG consisting of fludarabine 40 mg/m2 (D-8 to D-4), cyclophosphamide 60 mg/kg (D-3 to D-2) and total body irradiation (4 Gray) on D-1. Median age at time of UCBT was 14 (4-52) years. The median total nucleated cell number and CD34-positive cell number at infusion were 3.5 (1.07-7.87)× 107/kg and 1.7 (0.69-5.27) × 105/kg, respectively. Ciclosporin (CsA) and mycophenolate mofetil (MMF) was given to both groups as prophylaxis for graft versus host disease (GVHD).Results:Neutrophil recovery (>0.5×109/L) was observed in 11 patients of the ATG group and the median time to engraftment was 19 (13-35) days. The median time to platelet recovery (>20 × 109/L) was 40 (24-153) days. Primary graft failure was observed in seven patients. Only 1 out of 17 patients in the No-ATG group had primary graft failure. The median time to neutrophil engraftment was 17 (13-36) days. The median time to platelet engraftment was 31 (17-65) days. During follow-up, 7 patients died before 1 year due to non-engraftment (n=4), infection (n=2) and encephalorrhagia (n=1) in the ATG group. Four patients died in the No-ATG group due to infection (n=3) and IV grade acute GVHD in the skin and the intestinal tract (n=1, induced by discontinuing medicine). Furthermore, conditioning regimen without ATG shows even more superiority in patients refractory to immunosuppressive therapy with ATG and/or CsA (n=8), who had neutrophil engraftment completely within 20 days after cord blood infusion.Conclusion:UCBT after a FLU-CY-TBI conditioning regimen without ATG for SAA patients is better than that with ATG, especially for patients refractory to immunosuppressive therapy. Pediatric and adult SAA patients who are younger than 50 years old, lack of HLA-matched sibling donor and refractory to immunosuppressive therapy should consider UCBT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Influence de facteurs obstétricaux sur les caractéristiques des greffons de sang de cordon ombilical

ObjectivesThere is a correlation between the success of the cord blood transplant and the numbers of HSC found in the unit of cord blood donation. The purpose of this analysis is to identify obstetric factors that may influence the quality of a cord blood unit taken during delivery. MethodsA retrospective study was carried out at the Liège cord blood bank on a sample of 7.463 cord blood units collected between 2000 and 2016. Eight obstetric factors were analyzed in relation to two dependent variables; the total nucleated cells (TNC) and the volume of one unit of cord blood. The Welch test, the percentiles 25 and 75 and the non-parametric Mann-Whitney Wilcoxon test were used. ResultsSeveral obstetric parameters were found to influence (P<0.05) the quality of the cord blood unit are: cord clamping (>15 seconds), the use of oxytocin during labor, a more advanced gestational age (38–41 weeks), a higher birth weight (>3300 gr), and a higher weight of the placenta (>500 gr). A female newborn and the use of epidural, influence (P<0.05) the number of TNC but not the volume of the cord blood unit. ConclusionWe can conclude that the birth weight, the weight of the placenta, the gestational age and cord clamping are parameters that could be used by maternity hospitals to identify births that enable more voluminous grafts which are richer in total nucleated cells.

ОЦЕНКА ЭФФЕКТИВНОСТИ ЭКСТРАКТА HALOCYNTHIA AURANTIUM ПРИ ЭКСПЕРИМЕНТАЛЬНОМ ЛУЧЕВОМ ПОРАЖЕНИИ

Проведено исследование лечебного эффекта экстракта из морского гидробионта Halocynthia aurantium при радиационном воздействии. Экстракт асцидии (ЭА) содержит комплекс биологически активных веществ и обладает способностью повышать неспецифическую резистентность организма к действию ряда экстремальных повреждающих факторов.Объектом исследования являлись мыши BALB/с подвергнутые γ-облучению излучением малой мощности.Цель работы состояла в оценке лечебного влияния ЭА на пострадиационную динамику показателей периферической крови и клеточность костного мозга (доза 5 Гр) и на исход радиационного поражения (доза 13 Гр). ЭА животные получали ежедневно в течение всего эксперимента, начиная в день окончания облучения.Методика взятия материала и анализ элементов периферической крови и костного мозга стандартная. Показано, что в ранние сроки после облучения наблюдалось резкое снижение общего числа лейкоцитов периферической крови и ядросодержащих клеток в костном мозге. Ежедневное применение ЭА способствовало ускорению восстановления клеточного состава костного мозга и увеличению общего числа лейкоцитов и тромбоцитов в периферической крови.Можно заключить, что применение ЭА после радиационного воздействия позволило существенно повысить вероятность благоприятного исхода острой лучевой болезни при дозе облучения 5 Гр и облегчить ее течение.

The number of CD34+CD133+ hematopoietic stem cells residing in umbilical cord blood (UCB) units is not correlated with the numbers of total nucleated cells and CD34+ cells: a possible new indicator for quality evaluation of UCB units.

Umbilical cord blood transplantation (UCBT) is often associated with delayed neutrophil and platelet recovery. Engraftment failure is another major obstacle. Several factors influence these serious complications, including the numbers of total nucleated cells (TNCs) and CD34+ cells which have been used as reliable factors for selecting UCB units for transplantation. However, whether both factors are reliable indices of the hematopoietic stem cell (HSC) activity of UCB units remains unknown. To evaluate the quality of UCB units, we quantified the actual number of transplantable CD34+CD133+ HSCs (tHSCs) residing in UCB units. The number of tHSCs was not correlated with the numbers of TNCs or CD34+ cells. These results strongly suggest that neither factor reflects the numbers of tHSCs residing in UCB units. To validate the significance of the number of tHSCs, further analysis is required to determine whether the number of tHSCs residing in UCB units is useful as a new indicator for the quality assessment of UCB units.

365 - Enabling efficient ex vivo expansion of long-term human hematopoietic stem and progenitor cells for cell therapy

Umbilical-cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSPC) for transplantation into patients lacking a suitable HLA-matched donor. However, due to the limited cell dose in each cord blood unit, individuals >60 kg are restricted from the use of UCB-based therapy. Ex vivo expansion of UCB CD34+ cells is one strategy employed to increase the hematopoietic cell dosage. A major limitation of current systems used for the expansion of HSPC is that ex vivo culture leads to expansion and differentiation at the expense of the most primitive pluripotent long-term stem cells. This has limited the clinical application of ex vivo expanded HSPC, since short-term progenitor cells only provide transient protection, ultimately reducing the long-term positive health outcomes, increasing the duration of hospitalizations, and health care costs per patient. Development of a culture system that expands both short-term and long-term HSPC would facilitate immune protection during the early phase of recovery, and provides a suitable solution for transfusion-independent hematopoiesis. Therefore, we sought to develop a HSPC culture medium that enables the expansion of both long-term and short-term HSPC, while maintaining their functional properties. To this end, we conducted several iterative rounds of Design of Experiments (DOE) involving multifactorial analysis, and mathematical modeling methods. Definitive Screening DOEs allowed us to identify optimal combinations and concentrations of essential media components, small molecules, and growth factors. The performance of candidate HSPC expansion media were evaluated after 7 days of culture, with the following attributes assessed: (1) viability of cells; (2) numbers of total nucleated cells; (3) percentages and numbers of CD34+ cells; (4) percentages and numbers of CD34+CD90+CD45RA- cells; (5) expression of aldehyde dehydrogenase by expanded CD34+ cells; and (6) colony-forming unit (CFU) assays. As the transplantation of HSPC in immuno-deficient mice is the gold standard in determining whether the expanded cells are engraftable, we plan to conduct these studies with the lead candidate HSPC expansion medium. Taken together, we seek to highlight our design philosophy in HSPC culture media development. We believe that our efforts are critical for the successful utilization of hematopoietic stem cell transplants in translational cell therapies.

634 - The Effect of Multiple Unit, Autologous Blood Donation Prior to Bone Marrow Harvest on the Nucleated Red Blood Cell Component of the Total Nucleated Cell Number

634 - The Effect of Multiple Unit, Autologous Blood Donation Prior to Bone Marrow Harvest on the Nucleated Red Blood Cell Component of the Total Nucleated Cell Number

Ex Vivo Expansion of CD34+ CD90+ CD49f+ Hematopoietic Stem and Progenitor Cells from Non-Enriched Umbilical Cord Blood with Azole Compounds.

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+CD34+ cells in the PB and BM by day 21 compared to non‐expanded and cytokine expanded grafts. The C7 expanded grafts maintained long‐term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre‐culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376–393

Combined mild hypoxia and bone marrow mesenchymal stem cells improve expansion and HOXB4 gene expression of human cord blood CD34+ stem cells

Cord blood (CB) is a rich source of hematopoietic stem cells (HSC). It has been used successfully to treat a variety of hematological and non-hematological disorders. Beside the advantages of CB, its main disadvantages are the limited number of stem cells available for transplantation and delayed engraftment. Identifying strategies to enhance expansion and self-renewal of HSCs can improve transplantation efficiency. The aim of this study was to examine different culture conditions on ex vivo expansion and homeobox protein Hox-B4 gene (HOXB4) expression in cord blood CD34+ stem cells. Human cord blood CD34+ HSC were cultured in serum-free medium supplemented with cytokines with and without a feeder layer in normoxia (21% O2) and mild hypoxia (5% O2). At the end of 7 days of culture, the highest number of total nucleated cells (TNC), CD34+ cells, colony forming units (CFUs) and HOXB4 mRNA were observed in a co-culture of HSC with a bone marrow mesenchymal stem cell(MSC) feeder layer at 5% O2.We concluded that the combination of bone marrow (BM)-MSC and mild hypoxia (5% O2) not only improved HSC expansion but also enhanced HOXB4 gene expression as a self-renewal marker of HSC, and better mimicked the niche microenvironment conditions.

Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells.

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems.

Effects of extract of Urtica dioica L. (stinging nettle) on the immune response of rats with severe malnutrition.

Background. Severe malnutrition is the main cause of immunodeficiency in children under five years old in areas of economic depression in developing countries, and it is associated with generalized atrophy of lymphoid tissues. Urtica dioica L. is a medicinal herb with multiple pharmacological functions; it acts as modulator of the immune response, stimulating the proliferation of T lymphocytes in vitro. Objective: To determine the ability of U. dioica L. to modulate the immune response of malnourished rats in the first 21 days of life. Methods: Third-degree malnutrition during lactation was induced by food competition, and treatment with U. dioica L was tested. Day-old Wistar rats were randomly assigned to three groups: a) the well-nourished group, b) the untreated malnourished group and c) the malnourished group treated with 0.2 g/mL U. dioica L. on days 14, 16, 18 and 20. On day 21, animals were euthanized, and the thymus, spleen, bone marrow and blood were obtained. Lymphocyte subpopulations were determined in the thymus and leukocyte subpopulations in blood. The total number of nucleated bone marrow cells was determined, and the proliferative capability of spleen lymphocytes from malnourished rats treated with U. dioica L. was compared to that of other groups. Results: U. dioica L. decreased CD4-CD8- lymphocytes and increased CD4+CD8+ lymphocytes in the thymus, while in the blood, it increased CD4+ cells, monocytes and total T lymphocytes. The proliferation of spleen cells was stimulated in the malnourished rats, and a decrease in total nucleated bone marrow cells was observed. An increase in the red blood cell count, hemoglobin and white blood cell count was observed in malnourished rats treated with Urtica dioica L. (p≤0.05). Conclusion: U. dioica L. had immunomodulatory effects since it promoted the differentiation of CD4-CD8- T lymphocytes towards CD4+CD8+ T lymphocytes and counteracted anemia and leucopenia associated with severe malnutrition. Key words: Severe malnutrition, immunodeficiency, immunomodulatory activity, T lymphocytes, Urtica dioica L.

Evaluating the Genotoxicity Enhancement of the Antileukemic Drug 6-Mercaptopurin When Combined With Iraqi Nerium oleander and Narcissus tazetta Extracts in vivo

The present study was aimed to evaluate the genotoxicity of the aqueous extracts of Nerium oleander leaves and Narcissus tazetta bulbs each alone and together with the antileukemic drug 6-Mercaptopurin (6MP) in order to investigate the extracts ability to elevate the chemotherapeutic drug genotoxicity which may influence its treatment of cancer. The cytotoxicity test shows that the LD50 of the aqueous extract of Narcissus tazetta was 752.083 mg/kg and for Nerium oleander was 922.023 mg/kg. On the basis of the achieved LD50 values, the doses 92, 46 ,23 mg/kg of Nerium oleander and the doses 75, 37,18 mg/kg of Narcissus tazetta extracts were chosen, depending on chromosome aberrations, Micronuclei and Mitotic index as a powerful cytogenetic assays in bone marrow cells of Swiss albino male mice. The result indicated that Nerium oleander extract alone at the dose 92mg/kg induced significant effect on centromere break and ring chromosome comparing with the negative control (untreated mice) and significantly increased the mean values of chromatid gap and ring chromosome when compared with the positive control ( 6MP ). While only the dose 46 mg/kg and 23 mg/kg of N. oleander aqueous extracts significantly decreased mitotic index and when combined with 6MP it can enhance its antimitotic activity but not significantly. Moreover the extracts alone and when combined with 6MP did not significantly change the total number of red blood micro nucleated cells. For Narcissus tazetta extract, the three experimental doses alone lead to significant increase in chromosomal aberrations like: chromatid break with fragment, chromatid break without fragment, chromatid gap, centromeric break, ring chromosome and dicentric chromosome. While only the dose 75mg/kg had induced significant structural chromosomal abnormalities such as chromatid break with fragment, chromatid break without fragment centromeric break and ring chromosome, when combined with 6MP. The three doses of N. tazetta extract alone, had led to significant reduction in mitotic index compared with untreated control and also its combination with 6-MP significantly decreased the percentage of mitotic index. Moreover, only the doses 75 mg/kg and 37 mg/kg of N. tazetta extracts, when had given alone caused significant increase of the total micronucleated cells. While only the dose 75mg/kg of N. tazetta had induced significant frequency of the total micronucleated cell when combined with 6MP. In the present report, we attempted to establish that N.tazetta and Nerium oleander aquatic extracts enhance the genotoxicity and bioactivity induced by the antileukemic drug 6MP , thus preventing the development of cellular drug resistance which is a major problem that can face cancer patients using this drug .The current study serve the purpose of which is to search for local plants that may contribute to the establishment of novel supportive complementary and alternative medicine (CAM) during the chemotherapy of cancer in Iraq , Further studies are merited to explore this possibility.

The Exhaustion of Adult Hematopoietic Stem Cells in Ex Vivo Cultures Can Be Overcome by a Histone Deacetylase Inhibitor

Adult hematopoietic stem cells (HSCs) provide an attractive target for gene modification strategies for the correction of genetic blood disorders. A significant barrier to the translation of these strategies to the clinic has been the decline in numbers of fully functional HSCs associated with ex vivo culture. In order to create optimal conditions for gene modification, HSCs are typically cultured with cytokines to trigger their proliferation. Cytokine stimulation, however, induces stem cell differentiation, resulting in loss of long-term marrow repopulating potential. To overcome this barrier, we treated two sources of adult HSCs, G-CSF mobilized peripheral blood (mPB) and bone marrow (BM) CD34+ cells with valproic acid (VPA), a histone deacetylase inhibitor. This approach has previously been shown to promote the expansion of fully functional cord blood HSCs (Chaurasia et al. JCI, 2014).Cryopreserved mPB or BM CD34+ cells were thawed and cultured in serum-free media supplemented with stem cell factor, FLT-3 ligand, thrombopoietin and interleukin 3. The cells were primed with cytokines for 16 hours followed by addition of VPA (1mM) and incubation in the presence of VPA and cytokines for 7 days. The viability, number of total nucleated cells (TNC), CD34+ cells and frequency of various HSC subpopulations were determined shortly after thawing the primary cells (PCs) and at daily time points, in both VPA-treated cultures and cultures containing cytokines alone (control).The viability of mPB and BM cells treated with VPA for 7 days, each exceeded 90%. While not significantly affecting the expansion of TNC, VPA treatment maintained a higher percentage of CD34+ cells throughout the culture period, and led to a higher expansion of CD34+ cells compared to that observed in control cultures (29 vs 5.5-fold in mPB and 21 vs 5-fold in BM, p<0.0001). The CD34+ fraction was further assessed for the co-expression of CD90 and CD49f, which are known to enrich for long-term repopulating HSCs. While only a small percentage of CD34+ cells from either uncultured PCs or control cultures expressed CD90 and CD49f (range 1-10%), up to 70% and 60% of VPA-treated CD34+ cells expressed CD90 and CD49f, respectively, after 24-48 hours of exposure to VPA. This rapid increase in phenotypically defined HSCs occurred prior to cell proliferation, and is attributed to epigenetic reprogramming. The combined effect of epigenetic reprogramming and proliferation led to a significant expansion of CD34+CD90+ and CD34+CD90+CD49f+ cells in both mPB and BM, not observed in the control cultures (Table 1).Since the phenotype of cells expanded ex vivo does not always correlate with function, we used aldehyde dehydrogenase (ALDH) activity as a functional marker of HSCs. VPA-expanded mPB CD34+ cells showed significantly higher ALDH activity compared to PCs and controls, after both 3 and 7 days of incubation (Table 1). We have previously shown that epigenetic reprogramming induced by VPA is linked to mitochondrial remodeling and suppression of ROS (Papa et al. ASH 2016). We used the mitochondrial membrane potential dye TMRM to assess mitochondrial activity, and found a significant decrease in TMRM in VPA-treated cultures from mPB, as compared to control cultures, at 3 and 7 days (Table 1). This reduction in mitochondrial membrane potential is associated with primitive HSCs.To test their functional capacity, we transplanted the progeny of 2X105 mPB and BM CD34+ cells cultured with or without VPA, into sub lethally irradiated NSG mice. The degree of human cell chimerism in mouse bone marrows was determined 16 weeks later by human CD45 expression. While mice transplanted with grafts from the control cultures had limited or no human chimerism (range 0-1.4%), those transplanted with VPA-treated mPB grafts had up to 50% human chimerism (range 2-50%) (Table 1). The engrafted cells belonged to both myeloid and lymphoid lineages. Comparable results were observed using BM.Several groups have demonstrated the application of expansion methods adapted from cord blood to adult HSCs. These efforts met with successful albeit modest results, perhaps owing to a limited effect of the evaluated agents on adultHSCs. To our knowledge, the above studies represent the most robust demonstration of expansion of HSCs from adult sources, evident as early as after 3 days of culture. These studies strongly support the evaluation of VPA treatment in HSC gene modification strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment.

Objective:Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method.Materials and Methods:In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units.Results:Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant.Conclusion:In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.

Two versus three day upfront use of granulocyte-colony stimulating factor in healthy bone marrow donors for pediatric bone marrow transplantation

In order to decrease donors’ exposure to granulocyte-colony stimulating factor (G-CSF), we compared the effect of two versus three days of G-CSF priming on CD34+ yield in bone marrow (BM) harvest. Although the number of BM-CD34+ cells was higher in 3day G-CSF priming, we achieved the same number of CD34+ cells per recipient’s weight in 2day G-CSF priming group, too. In addition, the number of total nucleated cells (TNC) harvested from BM were similar with two or three day regimen. But mononuclear cells (MNC) of the BM graft was higher in the 3day G-CSF priming group. Similar to CD34+ cell numbers, BM harvest yielded similar TNC, and MNC numbers per kilogram of the recipient. We also found that, young donors (≤10year) had more peripheral blood MNC, bone marrow MNC and CD34+ cell numbers. Another interesting finding of this study was obtaining adequate number of peripheral blood stem cells for leukapheresis with three day G-CSF administration. Since engrafment times were also similar in two groups, we concluded that 2-days G-CSF priming was resulted in sufficient mobilization of BM stem cells.