scholarly journals Generation and Evaluation of Isogenic iPSC as a Source of Cell Replacement Therapies in Patients with Kearns Sayre Syndrome

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 568
Author(s):  
Glen Lester Sequiera ◽  
Abhay Srivastava ◽  
Keshav Narayan Alagarsamy ◽  
Cheryl Rockman-Greenberg ◽  
Sanjiv Dhingra
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Reactive Oxygen Species Generation ◽  
Energy Deficit ◽  
Patient Specific ◽  
Multisystem Disorder ◽  
Differentiation Potential ◽  
Cell Replacement ◽  
Mtdna Mutations ◽  
Kearns Sayre Syndrome

Kearns Sayre syndrome (KSS) is mitochondrial multisystem disorder with no proven effective treatment. The underlying cause for multisystem involvement is the energy deficit resulting from the load of mutant mitochondrial DNA (mtDNA), which manifests as loss of cells and tissue dysfunction. Therefore, functional organ or cellular replacement provides a promising avenue as a therapeutic option. Patient-specific induced pluripotent stem cells (iPSC) have become a handy tool to create personalized cell -based therapies. iPSC are capable of self-renewal, differentiation into all types of body cells including cardiomyocytes (CM) and neural progenitor cells (NPC). In KSS patients, mutations in mtDNA are largely found in the muscle tissue and are predominantly absent in the blood cells. Therefore, we conceptualized that peripheral blood mononuclear cells (PBMNC) from KSS patients can be reprogrammed to generate mutation free, patient specific iPSC lines that can be used as isogenic source of cell replacement therapies to treat affected organs. In the current study we generated iPSC lines from two female patients with clinical diagnosis of classic KSS. Our data demonstrate that iPSC from these KSS patients showed normal differentiation potential toward CM, NPC and fibroblasts without any mtDNA deletions over passages. Next, we also found that functional studies including ATP production, reactive oxygen species generation, lactate accumulation and mitochondrial membrane potential in iPSC, CM, NPC and fibroblasts of these KSS patients were not different from respective cells from healthy controls. PBMNCs from these KSS patients in the current study did not reproduce mtDNA mutations which were present in muscle biopsies. Furthermore, we demonstrate for the first time that this phenomenon provides opportunities to create isogenic mutation free iPSC with absent or very low level of expression of mtDNA deletion which can be banked for future cell replacement therapies in these patients as the disease progresses.

scholarly journals Morphology and morphometry of feline bone marrow-derived mesenchymal stem cells in culture

2014 ◽  
Vol 34 (11) ◽  
pp. 1127-1134 ◽  
Author(s):  
Bruno B. Maciel ◽  
Carmen L.K. Rebelatto ◽  
Paulo R.S. Brofman ◽  
Harald F.V. Brito ◽  
Lia F.L. Patricio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Mononuclear Cells ◽  
Average Length ◽  
Therapeutic Option ◽  
Future Application ◽  
Morphometric Study ◽  
Morphometric Characteristics ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x10(6)) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs.

scholarly journals CD14+ monocytes repress gamma globin expression at early stages of erythropoiesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Heshusius ◽  
Esther Heideveld ◽  
Marieke von Lindern ◽  
Emile van den Akker
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Globin Genes ◽  
Beta Globin ◽  
Hematopoietic Progenitors ◽  
Peripheral Blood Mononuclear ◽  
Temporal Regulation ◽  
Gamma Globin ◽  
Cd34 Cells ◽  
Adult Hemoglobin

AbstractIn β-hemoglobinopathies, reactivation of gamma- at the expense of beta-globin is a prominent therapeutic option. Expression of the globin genes is not strictly intrinsically regulated during erythropoiesis, supported by the observation that fetal erythroid cells switch to adult hemoglobin expression when injected in mice. We show cultured erythroblasts are a mix of HbA restrictive and HbA/HbF expressing cells and that the proportion of cells in the latter population depends on the starting material. Cultures started from CD34+ cells contain more HbA/HbF expressing cells compared to erythroblasts cultured from total peripheral blood mononuclear cells (PBMC). Depletion of CD14+ cells from PBMC resulted in higher HbF/HbA percentages. Conversely, CD34+ co-culture with CD14+ cells reduced the HbF/HbA population through cell–cell proximity, indicating that CD14+ actively repressed HbF expression in adult erythroid cultures. RNA-sequencing showed that HbA and HbA/HbF populations contain a limited number of differentially expressed genes, aside from HBG1/2. Co-culture of CD14+ cells with sorted uncommitted hematopoietic progenitors and CD34-CD36+ erythroblasts showed that hematopoietic progenitors prior to the hemoglobinized erythroid stages are more readily influenced by CD14+ cells to downregulate expression of HBG1/2, suggesting temporal regulation of these genes. This possibly provides a novel therapeutic avenue to develop β-hemoglobinopathies treatments.

Generation of 2.5D lung bud organoid from human induced pluripotent stem cell

2021 ◽  
pp. 1-14
Author(s):  
Xun Xu ◽  
Yan Nie ◽  
Weiwei Wang ◽  
Imran Ullah ◽  
Wing Tai Tung ◽  
...  
Keyword(s):  
Single Cell ◽  
Disease Modeling ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Homogeneous Distribution ◽  
Cell Seeding ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
Defined Culture

Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70%confluence (SC_70%_hom) or a clump seeding group with heterogeneously distributed cells at 90%confluence (CL_90%_het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.

scholarly journals Kearns-Sayre syndrome with facial and white matter extensive involvement: a (mitochondrial and nuclear gene related?) neurocristopathy?

2017 ◽  
Author(s):  
Agostino Berio ◽  
Attilia Piazzi ◽  
Carlo Enrico Traverso
Keyword(s):  
Nervous System ◽  
White Matter ◽  
Neural Crest Cell ◽  
Nuclear Gene ◽  
Mtdna Mutations ◽  
Kearns Sayre Syndrome ◽  
Mtdna Deletion ◽  
The Common ◽  
Crest Cell ◽  
Extensive Involvement

The Authors report on a patient with Kearns-Sayre syndrome, large mtDNA deletion (7/kb), facial abnormalities and severe central nervous system (CNS) white matter radiological features, commonly attributed to spongy alterations. The common origin from neural crest cell (NCC) of facial structures (cartilagineous, osseous, vascular and of the peripheral nervous system) and of peripheral glia and partially of the CNS white matter are underlined and the facial and glial abnormalities are attributed to the abnormal reproduction/migration of NCC. In this view, the CNS spongy alterations in KSS may be not only a dystrophic process (leukodystrophy) but also a dysplastic condition (leukodysplasia). The Authors hypothesize that the symptoms may be related to mtDNA mutations associated to NCC nuclear gene abnormality. SOX 10 gene may be a nuclear candidate gene, as reported in some case of Waardenburg IV syndrome.

Abstract 12600: Microrna From Atrial Tissue Harvested During Transseptal Puncture as a Predictor for Non-pv Trigger in Patients Undergoing Af Ablation: Preliminary Results From a Pilot Study

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Luigi Di Biase ◽  
Aaron Baker ◽  
Xue Yan ◽  
Jason Lee ◽  
Francesco Santoro ◽  
...  
Keyword(s):  
Therapeutic Option ◽  
Standard Procedure ◽  
Expression Patterns ◽  
Pulmonary Veins ◽  
Challenge Test ◽  
Inverse Correlation ◽  
Patient Specific ◽  
Af Ablation ◽  
Atrial Tissue ◽  
Nonparametric Correlation

Introduction: Catheter ablation of atrial fibrillation (AF) is the most valid therapeutic option to achieve rhythm control. Pulmonary veins (PV) are the most known trigger of AF, although recently we have become more aware of the importance of non-PV triggers. Expression of microRNA (miRNA) has been shown to be regulated in many cardiovascular disease. We sought to study expression patterns of miRNA in patients (pts) with AF undergoing ablation to facilitate their application as both diagnostic and prognostic markers. Methods: As part of the standard procedure for AF ablation a double transseptal sample of myocardial tissue is obtained via a transseptal needle. The small piece of atrial septal tissue can be retrieved from the needle as a result of piercing the atrial septum. MiRNA was hybridized to microarrays to determine relative levels of miRNAs in the samples. For a subset of the miRNAs we validated expression through quantitative real time PCR. All pts underwent PV-antrum and non-PV trigger ablation guided by isoproterenol challenge test. Results: Atrial tissue of 11 pts undergoing AF ablation has been utilized for MiRNA assessment. Mean age was 61.27 ± 10.5 years and 8 (72.7%) pts were male. Six (54.5 %) pts had paroxysmal AF. During the ablation non-PV triggers were detected in 8 (72.7 %) pts. Recurrence of AF occurred in 3(27.3 %) pts. Expression of miR-21, miR-26a and miR-29a was higher in pts with non-PV triggers, while miR-30c had lower expression in pts who had recurrence of atrial tachyarrhythmias. Spearman’s nonparametric correlation coefficient was calculated and miR-21, miR-26a, miR-29a were positively correlated with non-PV triggers (r = 0.58, p=0.06 for all three miRNAs), while miR-30c level had inverse correlation (r = (-) 0.78 %, p=0.005) with recurrence (Figure). Conclusions: Expression of miR-21, miR-26a, miR-29a correlates with the presence of non-PV triggers. This information could be clinically relevant in planning patient specific procedures.

Abstract 241: Direct Injection of Bone Marrow Mononuclear Cells Reversed Experimental Diabetic Neuropathy by Augmenting Neovascularization and Providing Angio-neurotrophic Cytokines

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hyongbum Kim ◽  
Yong Jin Choi ◽  
Jong-seon Park ◽  
Masaaki Ii ◽  
Marcy Silver ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Diabetic Neuropathy ◽  
Neurotrophic Factors ◽  
Mononuclear Cells ◽  
Motor Nerve ◽  
Therapeutic Option ◽  
Direct Injection ◽  
Saline Group ◽  
Vasa Nervorum ◽  
Sciatic Nerves

Background: Bone marrow (BM)-derived mononuclear cells (MNCs) have been shown to effectively treat ischemic cardiovascular diseases. Recent evidence suggested that diabetic neuropathy (DN) is causally related to impaired angio-vasculogenesis in vasa nervorum and deficiency of angiogenic and neurotrophic factors. Accordingly, we sought to investigate whether DN can be ameliorated by local injection of BM-derived MNCs. Methods and Results: A severe peripheral neuropathy, characterized by significant slowing of motor nerve conduction velocities (MCV) developed 12 wks after the induction of diabetes with streptozotocin in Fisher rats (vs normal rats; 46.6±2.6 vs 32.0±2.5 m/s, P < 0.05). These rats were randomly assigned to MNC or saline injection groups (n = 9, each group) and received either 5x106 MNCs or saline intramuscularly around the sciatic nerves. In the MNC group, MCV were significantly improved within 4 wks after treatment (MNC vs Saline, 41.9±3.2 vs 32.7±2.8 m/s, P < 0.01). Microvascular circulation of sciatic nerve, measured by laser Doppler perfusion imaging was markedly increased only in the MNC group. Capillary density at 4 wks was significantly higher in the MNC group than in the saline group (68±5.9 vs 37±4.4 /cross section, P < 0.01). Robust engraftment of MNCs were observed in sciatic nerves, which sustained over 4 wks. A fraction of engrafted MNCs expressed endothelial markers suggestive of transdifferentiation into endothelial cells in the vasa nervorum. Intriguingly, a large number of the engrafted MNCs are following the course of vasa nervorum in close proximity. Real-time RT-PCR on sciatic nerves revealed that the expression of angio-neurotrophic factors were significantly increased in the MNC group compared to the saline group: VEGF (2.1 fold), FGF-2 (2.4), eNOS (18.1), Brain-derived neurotrophic factor (35.1), IGF-1 (25.5) (all P < 0.05). The protein levels were well correlated with mRNA expression levels. Conclusion: Local transplantation of BM-derived MNCs could improve experimental DN by augmenting neovascularization and increasing angiogenic and neurotrophic factors in peripheral nerves. These findings suggest that BM-MNC transplantation may represent a novel therapeutic option for treating DN.

PRTX-100 Inhibits Platelet Phagocytosis In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1081-1081 ◽  
Author(s):  
Chris Yatko ◽  
Christopher Herrem ◽  
Samia Siddiqui ◽  
Victor S. Sloan
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Protein A ◽  
Flow Cytometric Analysis ◽  
Human Platelets ◽  
Staphylococcal Protein A ◽  
Human Monocytes ◽  
Peripheral Blood Mononuclear ◽  
Vitro Assay

Abstract Background: In idiopathic thrombocytopenic purpura (ITP), autoantibodies bind to platelets which are then phagocytosed by monocytes/macrophages and removed by the reticuloendothelial system. PRTX-100 (Staphylococcal protein A) is being investigated for the treatment of ITP. Objective: To assess the effect of PRTX-100 on phagocytosis of platelets in an in vitro assay. Methods: Human monocytes were isolated from whole blood peripheral blood mononuclear cells (PBMCs) by adherence and cultured for 6 days in RPMI + 5% human serum. 48 hours prior to phagocytosis assay, PRTX-100 was added at 250, 25, and 2.5ng/ml. Human platelets were labeled with a fluorescent (PerCP) lipophilic dye and opsonized with an antibody to MHC Class I (W632). 2×10−5 monocytes were co-cultured with 2×10−7 labeled platelets for 1 hour at 37 ° C. All conditions were performed in triplicate. After an hour, phycoerythrin (PE) labeled anti-CD61 antibody was added to assess surface bound platelets versus ingested platelets. Phagocytosis was determined by flow cytometric analysis. The monocyte population was gated upon by forward and side scatter properties, then verified by staining with CD14-FITC. Percent phagocytosis was calculated as the fraction of ingested platelets (PerCP +/CD61−) to the total PerCP population (PerCP +/CD61−) + ( PerCP+/CD61+) within the gated monocyte population. Results: PRTX-100 inhibits the phagocytosis of W632 opsonized platelets by human monocytes. Phagocytosis of W632 opsonized platelets was 40%, while phagocytosis in the presence of PRTX-100 at concentrations of 250, 25, and 2.5ng/ml was 18.3%, 23%, and 24.3%, respectively. Phagocytosis at 250ng/ml and 25ng/ml was significantly different from control phagocytosis with p values of 0.014 and 0.001 respectively by Student’s t test. Conclusions: PRTX-100 inhibits the phagocytosis of platelets by monocytes, the effector limb of ITP. Prevention of platelet phagocytosis is an important treatment goal in ITP. PRTX-100 has been shown to be generally safe and well-tolerated in a phase I study in healthy volunteers (J Clin Pharmacol, in press). PRTX -100 is a promising therapeutic option for ITP and deserves further study. Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets

GMP-Conform Generation and Cultivation of Unrestricted Somatic Stems Cells (USSC) from Cord Blood Using the SEPAX©-Separation Method a Closed Culture System Applying Cell Stacks.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1211-1211 ◽  
Author(s):  
Teja F. Radke ◽  
Anja Buchheiser ◽  
Aurélie Lefort ◽  
Mahtab Maleki ◽  
Peter Wernet ◽  
...  
Keyword(s):  
Cord Blood ◽  
Tissue Regeneration ◽  
Closed System ◽  
Tissue Repair ◽  
Mononuclear Cells ◽  
Calf Serum ◽  
Processing System ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Laboratory Conditions

Abstract Generation and characterization of unrestricted somatic stem cells (USSC) from cord blood (CB) was described by our group and has been well established under laboratory conditions [Koegler et al 2004, 2005 and 2006; Sensken et al 2007]. Due to their proliferative and differentiation capacity, USSCs are an interesting candidate for the future development of cellular therapy for tissue repair and tissue regeneration as well as a supportive cell layer to support hematopoietic reconstitution. Since generation and expansion under GMP-grade conditions is mandatory for use in clinical application, the automated cell processing system Sepax (BIOSAFE) with the CS900 separation kit was used for mononuclear cell separation and the subsequent generation of the USSC colonies in the presence of 30% GMP-grade fetal calf serum (Perbio), low-glucose DMEM-medium/10-7M dexamethasone. Expansion of USSC was performed in a closed system (Macopharma) applying cell stacks (Costar Corning). Results achieved so far indicate that the generation frequency and quality of generated USSC under GMP conditions are equal or even superior (45%) to manual generation under laboratory conditions (43%). 20 cord-blood units (mean volume 88,5 +− 15,8 ml; mean number of mononuclear cells 3,1 +−0,6 *108 MNC) have been processed, resulting in 9 USSC-colony formations and lines within 14–28 days. Growth kinetics is equal to the previously established USSC-lines (∼36–48 h / population doubling). Analysis of the immunophenotype as well as the differentiation potential towards the mesenchymal, neural and endodermal lineages also showed no difference to those lines generated manually using Ficoll-separation and normal cell culture flasks (Costar Corning T225). The closed system applied here is perfectly suitable to ensure safe and easy handling of the USSC, including seeding, trypsination and harvesting. In combination with the cell stack system (1, 2, 5 and 10 layers), cell amounts of more than 1.0×109 USSC can be achieved within 4 passages. These USSC products were temperature controlled cryopreserved in the presence of 10% DMSO, HSA and dextran. USSC can be thawed and further expanded in clinical grade quality. On the basis of their pluripotency and expansion under GMP-conditions into large quantities, these USSC from cord blood, when pretested for infectious agents and matched for the major transplantation antigens, may serve as a universal allogeneic stem cell source for tissue repair and tissue regeneration.

Human Erythroblasts Generated in Vitro Remain Functional with a Normal Karyotype 8 Years after Cryopreservation: Implications for Ex Vivo Generated Erythroid Transfusion Products.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2303-2303 ◽  
Author(s):  
Massimo Sanchez ◽  
Amanda Leblanc ◽  
Annalisa Mancini ◽  
Francesca Masiello ◽  
Valentina Tirelli ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Blood Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Genomic Rearrangements ◽  
Multicolor Fish ◽  
Peripheral Blood Mononuclear ◽  
Differentiation Potential ◽  
Blood Mononuclear Cells

Abstract The safety and adequacy of the blood supply is threatened by natural disasters, social and political events, epidemics, and emerging infections. During shortages, frozen blood is used to supplement the blood supply. Current regulations allow red blood cells to be stored frozen up to ten years; however, the shelf-life of such products is limited once blood is thawed. Cultured human erythroid cells derived in vitro from either fresh or cryopreserved CD34+ cells or peripheral blood mononuclear cells potentially represent an alternative source of erythrocytes for transfusion. However, it is unknown if normal erythroid cells undergoing ex-vivo expansion with growth factors will remain functional or develop genetic rearrangements in culture making them unsuitable for transfusion. We have compared the proliferative and differentiation potential of human erythroblasts obtained in culture from the peripheral blood mononuclear cells (PBMC) of adult donors. This analysis included freshly expanded erythroblasts as well as erythroblasts cryopreserved and stored for short (1 month) and long (8 years) periods. PBMC from four volunteer blood donors were prepared using gradient-density centrifugation and cryopreserved in DMSO in June 2000. One months later, 2x107 PBMC from one of the donors were thawed and cultured under conditions that allow massive ex vivo generation of erythroblasts (HEMA culture, Migliaccio et al Blood Cells Mol Dis2002;28:169-80). These cultures were stimulated with recombinant hSCF (50ng/mL), hGM-CSF (1ng/ml), hIL3 (1U/mL), hEPO (1U/mL) and contained dexamethasone and estradiol (each 10−6 M). Twenty million PBMC from the three additional donors were thawed and cultured under HEMA conditions in 2008. In all the three cases, the day 9 cultures contained an average of 10x107 cells, 95% of which were erythroid by CD36 and CD235a staining. These day 9 cells were either cultured for 4 additional days or cryopreserved (&gt;10 individual vials per donor containing 5x106 each). Cells were subcultured and maintained either under HEMA conditions (to assess their proliferation ability) or stimulated with EPO alone (5U/ mL) (to assess maturation). In May 2008, aliquots of the erythroblasts obtained from all donors were thawed and cultured again and amplification and differentiation potential of the freshly expanded and thawed cells were compared. Cells thawed after few months or 8 years of cryopreservation gave similar results and the data were pooled. The viability of the erythroblasts after thawing was 60–70%. After 4 days under HEMA conditions, both freshly expanded and cryopreserved erythroblasts doubled in numbers and retained an immature erythroid phenotype (CD36highCD235alow). On the other hand, in cultures containing EPO alone, the erythroblasts remained constant in number but progressed to a mature CD36posCD235ahigh phenotype. The results are summarized in the following table: Proliferation and Maturation Profile of Fresh and Cryopreserved Human Erythroblasts Fold Increase Phenotype CD36highCD235alow CD36highCD235ahigh Fresh cells HEMA culture 2 53% 40% EPO alone 1 15% 80% Thawed Cells HEMA culture 2 46% 36% EPO alone 1 5% 90% The eight-years cryopreserved erythroblasts expanded in culture were also cytogenetically evaluated. Karyotype and multicolor FISH analyses demonstrated a normal 46,XY karyotype with no obvious genomic rearrangements. To determine whether cells carried any known in utero leukemic genomic rearrangements, interphase FISH studies were performed for TEL/ETV6-AML1, MLL, 5q31 (EGR1) and 7q31 loci. In 800 evaluated interphase nuclei, all loci were present in disomy. This data indicates that human erythroblasts obtained in culture can be efficiently cryopreserved, remain functional in culture and do not acquire chromosomal abnormalities detectable by multicolor FISH analysis. These observations suggest that cultured erythroblasts should be further evaluated to determine if they represent a more suitable long term storage product than cryopreserved mature red blood cells.

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