Increasing Post-Processing Total Nucleated Cell (TNC) Recovery in Cord Blood Banking: Hespan Addition in cGMP Environment

Abstract Purpose. Evaluate the effects of HESPAN (HES) addition on indices of cord blood unit (CBU) potency, stability and safety after automated volume reduction (reduction of erythrocyte bulk and plasma volume) using the AXP AutoXpressTM (AXP) processing system. Background and Methods. TNC recovery varies significantly and unpredictably after volume reduction during CBU processing. However, prompt engraftment of CBU allotransplants correlates with their TNC including hematopoietic stem/progenitor cells (HPC). As a result, TNC is important in selecting CBU for transplantation: for example, 75% of the National Cord Blood Program (NCBP) CBUs shipped during the period 2010-2013 had TNC >120 x 107. Therefore, minimizing TNC losses during processing improves the chances that a CBU will be useful for clinical transplantation. The first method to increase post-processing TNC recovery was the addition of HES to the centrifugal method for CB volume reduction [Rubinstein et al, PNAS (USA), 92:10119-10122, 1995]. Volume reduction consists of concentrating the buffy-coat (containing leukocytes and HPC) by centrifugal stratification and removal of bulk red cells and platelet-containing plasma. NCBP processed manually over 30,000 CBUs using HES during the period 1995-2006. In 2006, the AXP, a closed, automated and FDA-approved processing method, without HES, was implemented. AXP was designed to enhance the MNC and CD34+ cell recoveries but not those of granulocytes. This resulted in lower post- processing TNC counts. Additionally, CBUs with larger volume and TNC content have somewhat higher TNC losses during automated processing. Using the AXP method, NCBP’s HEMACORD® obtained FDA License approval on November 10, 2011. However, since TNC (not MNC or CD34+ count) remains the most commonly used indicator of CBU potency and engraftment ability, we describe here implementation of HES in AXP processing to augment TNC recovery, by adding HES to the CBU to a 1-2% final concentration. Results.The results of manual CBU processing showed that HES addition improves yield, without changes in cell viability after cryopreservation, freezing at -196°C and thawing, after 20 years (from NCBP continuing stability study). HES addition also preserved CFU numbers and CD34+ cell counts after thawing. 1. Comparison of TNC recoveries without and with HES addition in the same CBU: A total of 25 CBUs were initially processed with the AXP platform without HES, as per routine procedure, and the TNC recoveries were calculated. Each CBU was then reconstituted after its initial processing into a new AXP bag set. HES was added aseptically to the reconstituted product and each CBU was processed in the same conditions as first time (same AXP device, centrifuge, etc.). The TNC recoveries after the second volume reduction process (AXP with HES) were ~20% higher on average than after the first (AXP without HES). 2. Comparison of TNC recoveries in different cohorts of CBUs: Thirty clinical-grade CBUs, with volumes 80-156 mL and TNC counts 111-290 x 107, were processed with HES in the AXP system and the results were compared with those of AXP-processed CBUs without HES over an earlier six month period. TNC and CD45+ cell recoveries improved by 16 - 20% maintaining mean post-processing hematocrit at 30.6% (SD ± 2). CD34+ and CD45+ cell viabilities were unchanged: 99% (SD ± 0.7) and 96% (SD ± 2.7), respectively, while their mean recoveries were 95% (SD ± 18) and 94% (SD ± 6). In addition, the same consistent post-processing volume was obtained (mean 20.8 mL, SD ± 0.1). CFU - CD34+ correlation (R2) after processing with HES was 0.788 (not different than what was observed in the CBUs without HES). Six CBUs AXP-processed with HES, were thawed and tested, with very minor losses in cell count and viability, similar to results of thawed AXP-processed CBU without HES. Finally, HES addition did not result in microbial contamination in any of these AXP-processed CBUs. Conclusion. Adding HES to CBUs before automated AXP processing increased substantially the TNC and CD45+ recoveries without loss of viability, while the CD34+ recoveries remained basically unchanged, with a mean of 95%. The post-processing hematocrit was consistent and low. AXP-automated CB processing with HES addition can be performed in the GMP environment, results in higher post-processing TNC and therefore, increases the CB bank’s ability to store larger CBUs that are most useful to patients. Disclosures No relevant conflicts of interest to declare.

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Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation

10.1101/181057 ◽

2017 ◽

Author(s):

John Dou ◽

Rebecca J. Schmidt ◽

Kelly S. Benke ◽

Craig Newschaffer ◽

Irva Hertz-Picciotto ◽

...

Keyword(s):

Dna Methylation ◽

Blood Cell ◽

Cord Blood ◽

Whole Blood ◽

Cell Types ◽

Buffy Coat ◽

Sample Type ◽

Blood Samples ◽

Cell Composition ◽

Cell Counts

AbstractBackgroundCord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells by generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability.ObjectivesTo evaluate differences in cell composition and DNA methylation between buffy coat and whole cord blood samples.MethodsCord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in 8 individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition.ResultsDNA methylation PCs were associated with individual (PPC1=1.4x10-9; PPC2=2.9x10-5; PPC3=3.8x10-5; PPC4=4.2x10-6; PPC5=9.9x10-13), and not with sample type (PPC1-5>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired individual samples ranged from r=0.66 to r=0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (5 sites had unadjusted P<10-5). Estimated cell type proportions did not differ by sample type (P=0.86), and estimated cell counts were highly correlated between paired samples (r=0.99).ConclusionsDifferences in methylation and cell composition between buffy coat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.

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Routine Automated Processing of Cord Blood Units Using the Sepax Cell Processing System.

Blood ◽

10.1182/blood.v108.11.3646.3646 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3646-3646

Author(s):

Safa Karandish ◽

Nery Berrios ◽

Sufira Kiran ◽

Charisse Ayuste ◽

Toby Hamblin ◽

...

Keyword(s):

Cord Blood ◽

Processing Time ◽

Processing System ◽

Buffy Coat ◽

Bar Code ◽

Cell Processing ◽

Wide Range ◽

Significant Difference ◽

Centrifugation Step ◽

Cryoprotectant Solution

Abstract We recently incorporated the use of the automated Sepax Cell Processing System (Biosafe SA, Switzerland) for red cell and volume reduction of cord blood units (CBU) before cryopreservation. Now that we have been routinely using this new technique in the laboratory for about six months, we decided to compare the results of this method with our standard manual processing method (Rubinstein, et al, PNAS1995,; 92: 10119–22). For both methods, hespan is added to the cells at final concentration of 20% (v/v). With the Sepax system, after addition of hespan, the cell bag is connected to the Sepax tubing set with the final freeze bag pre-attached to the set. After completion of the automated procedure, buffy coat is collected in the final freeze bag. Cryoprotectant solution is then added directly to the freeze bag. In the manual method, buffy coat and white cell rich plasma layer is collected after first centrifugation step and the white cells are separated from the plasma after the second centrifugation step. Cryoprotectant solution is then added to the cells before transfer to the final freeze bag for cryopreservation. The following are summary of results for each method: Table 1 Manual (n=1160) Sepax (n=311) Pre-Processing volume (ml) 107±30 114±28 Pre-Processing TNC (×10e6) 1196±577 1315±519 TNC Recovery (%) 80±8 83±8 TNC Viability (%) (7-AAD) 97±3 98±3 Total CD34 (×10e6) 4.3±4 4.9±3.6 Total DFU (×10e6) 70±0.9 61.5±20 Post Processing RBC Volume (ml) 9±2 7.3±2 Total Processing Time (including Setup) 60 minutes 30 minutes It is important to note that there was not a significant difference in TNC Recovery over a wide range of Pre-Processing Volume (66–206ml) or Pre-Processing TNC (440 – 3559×106). Since the Sepax device is an automated procedure, issues could arise (i.e. short term loss of electrical power) that would require us to reprocess the CBU before cryopreservation. The Sepax system allows for recovery and reprocessing of the cell using the ‘Purge mode’. We used 5 CBU units to evaluate TNC recovery and viability after purging the cells once and reprocessing. Table 2 TNC Recovery (%) TNC Viability (%) First Buffy Coat 85±5 98±0.9 Post Purge and reprocessing 76±5 98±0.9 Although the TNC recovery was lower after the second procedure, it was still within acceptable limits and the viability of the cells had not changed. These data demonstrates that both methods are equivalent with respect to cell recovery. However, the Sepax System substantially reduces processing time and hands-on operator intervention. Additionally system provides, closed-system processing, bar code reading capability and run data print-out suitable for GMP manufacturing settings.

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HHV-6 Encephalitis in Pediatric Unrelated Umbilical Cord Blood Transplantation: Role for Ganciclovir Prophylaxis?

Blood ◽

10.1182/blood.v114.22.4667.4667 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4667-4667

Author(s):

Frankie Wai Tsoi Cheng ◽

Vincent Lee ◽

Wing Kwan Leung ◽

Paul Kay Sheung Chan ◽

Ting Fan Leung ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Conflicts Of Interest ◽

Rare Complication ◽

Cord Blood Transplantation ◽

The Other ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Before And After

Abstract Abstract 4667 Background The role of ganciclovir as HHV-6 prophylaxis in unrelated hematopoietic stem cell transplant (HSCT) setting remains controversial. Methods We performed a 8-year retrospective review of patients received unrelated HSCT from January 2000 to September 2008. From January 2002, ganciclovir prophylaxis 5mg/kg twice daily for 7 days for all unrelated HSCT before transplant was adopted. The other transplant policies including antibacterial, antifungal, antiviral and graft-versus-host disease control policies remained unchange in that period. The prevalence of HHV-6 encephalitis was studied before and after the change in policy. Result Fifty-four unrelated HSCT were performed from January, 2000 to September, 2008. Total four cases (7.4%) of HHV-6 encephalitis were diagnosed. Two cases out of 16 cases (12.5%) diagnosed before adoption of the policy; 2 cases out of 38 cases (5.3%) diagnosed afterward. All of them were unrelated umbilical cord blood (UCB) transplant recipients. Two cases had significant residual neurological deficit and refractory seizure. The other two cases died of other transplant-related mortalities. Conclusion We conclude that HHV-6 encephalitis is still a rare complication of unrelated HSCT and may be more common in unrelated UCB transplantation. Routine use of ganciclovir as HHV-6 prophylaxis in all unrelated HSCT recipients may not be justified. Disclosures: No relevant conflicts of interest to declare.

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Characterization of the Putative Stem Cells From Umbilical Cord Blood.

Blood ◽

10.1182/blood.v114.22.4231.4231 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4231-4231

Author(s):

Amos S. Gaikwad ◽

Michael Cubbage ◽

Tatiana Goltsova ◽

Christopher Threeton ◽

Maria Ty ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cord Blood ◽

Cell Population ◽

Cell Biology ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Side Population ◽

Hematopoietic Stem ◽

Cell Fractions

Abstract Abstract 4231 Cord blood (CB) is a rich source of hematopoietic stem cells (HSC) with long-term repopulating activity necessary for allogeneic stem cell transplantation. CD34+ stem cells are considered sufficient for transplantation, however recent progress in stem cell biology indicates that cells with other surface markers such as CD133 or cells expressing high aldehyde dehydrogenase activity with low side scatter (ALDHhigh/SSClow) or a rare side population (SP) of cells that exclude the Hoechst 33342 vital dye via multi drug transporters have been shown to possess stem cell properties. We characterized CD34+, CD133+, ALDH+ and SP in mononuclear cells (MNC) isolated from human CB. While the SP cell population is rare and detectable in few CB-MNC examined, we found abundant CD34+ and CD133+ cells (1.0+/-0.5 and 0.8+/-0.4 per 100 CD45+ MNC cells, respectively) following the ISHAGE protocol. A distinct ALDH+ cell population (median of 0.26%; range of 0.1 to 0.5%) was also present in all of the CB-MNC analyzed. Over 90% of the ALDH+ cells were also CD34+ and CD133+. The ability of CB-MNC to form colonies in methocult semi-solid media supplemented with cytokines yielded myeloid, lymphoid and erythroid colonies. The clonogenic potential of CB-MNC ranged from 16-48%. We are assessing the colony forming ability of purified stem cell fractions using flow cytometry. The clonogenic efficiency of these individual putative stem cells will be discussed. Disclosures: No relevant conflicts of interest to declare.

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Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽

10.1182/blood.v116.21.3722.3722 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3722-3722

Author(s):

Li Ming Ong ◽

Xiubo Fan ◽

Pak Yan Chu ◽

Florence Gay ◽

Justina Ang ◽

...

Keyword(s):

Cord Blood ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Insulin Growth Factor ◽

Nsg Mice ◽

Nonobese Diabetic ◽

Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

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“RUNX1-IRES-GFP Knock-in” Mice Lack Expression of Runx1a: A Versatile Tool for Hematopoietic Stem Cell Analysis

Blood ◽

10.1182/blood.v118.21.2329.2329 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2329-2329

Author(s):

Yukiko Komeno ◽

Ming Yan ◽

Shinobu Matsuura ◽

Miao-Chia Lo ◽

James R. Downing ◽

...

Keyword(s):

Body Weight ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Hematopoietic Stem ◽

Blood Indices ◽

Cell Counts ◽

Significant Difference ◽

Versatile Tool ◽

Exon 4 ◽

Runx1 Expression

Abstract Abstract 2329 Previously reported “RUNX1-IRES-GFP knock-in mice” (Blood 2004;103:2522) (KI mice) were generated by replacing exon 4 of runx1 gene with cDNA of Runx1b/c from exon 4 to exon 8 followed by IRES-GFP, aiming to evaluate Runx1 expression in specific lineages and developmental stages during adult hematopoiesis. They are phenotypically normal, fertile, and blood indices are normal. GFP intensity correlates with Runx1 expression level, and shows lineage-specific changes during maturation in myeloid, erythroid, and lymphoid cells. However, the behavior in the hematopoietic stem cells (HSCs) had not been carefully examined. Interestingly, we discovered that this knock-in strategy eliminated Runx1a expression. Since Runx1a expression is relatively higher in HSCs than in differentiated cells, we analyzed HSCs in these mice to evaluate its roles in stable and stress hematopoiesis. We found that LSK fraction in bone marrow (BM) was significantly decreased in KI mice compared to wild type (WT) mice (0.043% vs 0.085%, p = 0.001). Among subpopulations in LSK, short-term HSC and multipotent progenitor fractions were significantly decreased (0.024% vs 0.046%, p = 0.003, 0.0021% vs 0.0026%, p = 0.001, respectively). SLAM marker staining using CD150 and CD48 showed similar results. Competitive repopulation assay showed less functional HSCs in KI mice. However, there was no significant difference in recovery of cell counts after single-dose 5-FU intraperitoneal injection (150 mg/kg body weight) or sublethal irradiation (5 Gy), or survival after weekly 5-FU injection. After G-CSF subcutaneous injection (125 μg/kg body weight, twice daily for 5 days), mobilized WBC or neutrophil in PB showed no difference. However, LSK and long-term HSC in PB were significantly less in KI mice (0.078% vs 0.135%, p = 0.010, 0.043% vs 0.092%, p = 0.029, respectively) while those in BM did not show significant difference (increased to 0.295% and 0.346% in KI and WT mice, respectively). In conclusion, Runx1a plays some non-redundant roles in stable hematopoiesis, while it is dispensable for tested stress hematopoiesis. RUNX1-GFP KI mice are a versatile tool to evaluate roles of Runx1a in normal hematopoiesis and leukemogenesis when combined with other genetic modifications. Disclosures: No relevant conflicts of interest to declare.

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Hhex Is a Critical Gene In The Development Of Normal and Malignant Lymphoid Cells

Blood ◽

10.1182/blood.v122.21.3788.3788 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3788-3788

Author(s):

Charnise Goodings ◽

Stephen B. Smith ◽

Elizabeth Mathias ◽

Elizabeth Smith ◽

Rati Tripathi ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Transgenic Mice ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Conditional Knockout ◽

Hematopoietic Stem ◽

Direct Transcriptional Target ◽

Cell Counts ◽

Transcriptional Target

Abstract Hematopoietically expressed homeobox (Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEX is a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEX clusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice (figure 1). Hhex conditional knockouts (Hhex cKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.Figure 1Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic miceFigure 1. Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice Disclosures: No relevant conflicts of interest to declare.

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Defining Parameters That Can Guide Public Cord Blood Units (CBUs) in Korea

Blood ◽

10.1182/blood.v126.23.3102.3102 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3102-3102

Author(s):

Junglim Lee ◽

Byung-Su Kim ◽

Soon- Hi Jang ◽

Tai-Gyu KIM

Keyword(s):

Multivariate Analysis ◽

Cord Blood ◽

Umbilical Cord ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Female Gender ◽

Limiting Factor ◽

Study Cohort ◽

Normal Delivery ◽

Hematopoietic Stem

Abstract Background: It is well known that Total Nucleated Cell (TNC) and CD34 are the greatest limiting factor in the use of umbilical cord blood (UCB) for transplantation. To enhance high UCB quality, it is important that how the factors will affect the UCB. This study is to identify maternal, neonatal, obstetric factors that influence the suitability for banking and transplantation of UCB units collection. Study design and methods: This study examined 6534 UCB (3712 at the Catholic Hematopoietic Stem Cell Bank and 2822 at the Daegu Fatima Hospital Public Umbilical Cord Bank) collected at two banks from October 2003 to June 2015. The variables were collected from retrospective records at the time of donation. The associations between TNC, CD34+ and variables including maternal age (MA), gestational age (GA), fetal body weight (FBW), time from collection to processing (T), collecting volume (CV), preTNC, and delivery type were analyzed by logistic regression. Results: In our study cohort (n=6534, male 2988, female 2991, unkown 555, all Koreans), the median values of TNC, numbers of CD34+, MA, GA, FBW, T, preTNC, and CV were 9.24úI108/unit (range, 3.02-35.64), 2.0úI106/unit(range, 0.04-29.2), 31.0 years(range, 15-46), 277 days (range, 202-382), 3330g (range, 1740-4970), 19 hours (range, 1-54), 11.69úI108/unit (range, 3.41-50.32), and 83.5ml (range, 26.0-218.2) respectively. In univariate analysis, variables that were associated with high TNC (defined as a TNC of > 9.24úI108/unit) included GA (defined as GA > 277 days) [OR 1.29 (95% CI 1.16-1.42 p < 0.001)], FBW (defined as FBW > 3330g) [OR 1.52 (95% CI 1.37-1.68 p < 0.001)])], CV (defined as > 83.5mL) [OR 2.61 (95% CI 2.36-2.88 p < 0.001)], preTNC [OR 25.45 (95% CI 22.34-29.00 p < 0.001)], and T (defined as T> 19 hours) [OR 0.87 (95% CI 0.79-0.96 p < 0.005)]. Variables that were associated with high CD34+ (defined as a number of CD34+ > 2.0úI106/unit) included MA (defined as MA > 31.0 years) [OR 0.90 (95% CI 0.82-0.99 P=0.036)], GA [OR 0.74 (95% CI 0.67-0.82 p < 0.001)], FBW [OR 1.41 (95% CI 1.27-1.56 p<0.001)], preTNC [OR 3.38 95% CI 3.06-3.74 p < 0.001]], and CV [OR 1.41 (95% CI 1.28-1.60 p<0.001)] In multivariate analysis of TNC, preTNC [OR 20.71 (95% CI 17.87-24.00) p < 0.001]] was the best predictor of followed by normal delivery [OR 1.77 (95% CI 1.48-2.11 P<0.001)], FBW [OR 1.35 (95% CI 1.17-1.56 p<0.001)], CV [OR 1.31 (95% CI 1.13-1.53 p<0.001)], and female gender [OR 1.21 (95% CI 1.05-1.39 p=0.01)]. In multivariate analysis of CD34, was preTNC [OR 3.39 (95% CI 3.00-3.83 p < 0.001)] was the best predictor of followed by FBW [OR 1.41 (95% CI 1.25-1.58 P<0.001)], GA [OR 0.59 (95% CI 0.52-0.66 p<0.001)], MA [OR 0.84 (95% CI 0.75-0.94 p=0.003)], and female gender [OR 0.89 (95% CI 0.78-0.98 p=0.02)]. Conclusions: We established referential values of cord blood using large scaled CB units in Korea. In multivariate analysis, maternal/donor characteristics were associated with preTNC, FBW, and gender for both high TNC and CD34+. Our results confirm that is similar values to those reported in previous data. These associations could be used to prioritize donations, collections, optimizing resource utilization and financial modeling in Korean cord blood banks. We are focusing on collection education using the standard operation procedure to facilitate of high cells as well as on more recruits of healthy mothers. Disclosures No relevant conflicts of interest to declare.

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SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2021-153814 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2189-2189

Author(s):

Mark C Wilkes ◽

Aya Shibuya ◽

Vanessa M Scanlon ◽

Hee-Don Chae ◽

Anupama Narla ◽

...

Keyword(s):

Cord Blood ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Culture Media ◽

Hsp70 Expression ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Enhancer Element ◽

Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

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IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Blood ◽

10.1182/blood-2020-137026 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Yang Mei ◽

Yijie Liu ◽

Xu Han ◽

Jing Yang ◽

Peng Ji

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Inflammatory Cytokines ◽

Conflicts Of Interest ◽

Double Knockout ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Cell Counts ◽

Age Related

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

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