Articles de revues : « StemCell »

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Brower, Vicki. « CT turns focus to StemCell ». Nature Biotechnology 17, n^o 9 (septembre 1999) : 838. http://dx.doi.org/10.1038/12794.

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Ciment, J. « US Congress debates stemcell research ». BMJ 318, n^o 7178 (23 janvier 1999) : 215. http://dx.doi.org/10.1136/bmj.318.7178.215b.

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De Oliveira, Cristiana Dias, Sudivan Vieira, Flávia dos Santos Lugão De Souza, Francimar Tinoco De Oliveira, Valéria Zadra De Mattos, Ana Lúcia Cascardo Marins et Stelmar Mollas Moura. « Stemcell therapy for cardiology : a new path to humanity and a new care for nurse - a case report ». Online Brazilian Journal of Nursing 4, n^o 1 (18 avril 2005) : 2–8. http://dx.doi.org/10.17665/1676-4285.20054783.

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During the process of care of patients who suffered acute myocardial infarction we observed how complex is this disease. Nowadays, stemcell therapy opens a new horizon and hope for these patients. We conducted this study to improve our knowledge as nurses in face of this new therapeutic model. This study is a case report of a patient who suffered an anterior acute myocardial infarction and was admitted in a coronary care unit and enrolled in stemcell transplantation study protocol. The nurse has a fundamental role in the process of care during the hospitalization period clarifying the patient’s doubts and perspectives. The data was collected in patient’s record.During the patient length of stay we emphasized our nursing care based on his individual value.

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Mhashilkar, Abner, et Anthony Atala. « Editorial : Effective Bio-EconomicApproaches for StemCell Therapy and Regenerative Medicine ». Current Stem Cell Research & ; Therapy 7, n^o 1 (1 janvier 2012) : 1. http://dx.doi.org/10.2174/157488812798483430.

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Stephens, M. « 1146 Quality of life after stemcell transplantation — a qualitative study ». European Journal of Cancer Supplements 1, n^o 5 (septembre 2003) : S348. http://dx.doi.org/10.1016/s1359-6349(03)91172-4.

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Saha, Partha, et Arunima Biswas. « Non-Canonical WNT Pathway in Breast Cancer and Breast Cancer StemCell Signalling ». Journal of PharmaSciTech 9, n^o 1 (1 septembre 2020) : 22–27. http://dx.doi.org/10.33981/jpst.2020.v09i01.004.

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Duerken, Matthias, Henriette Schneider, Jan Soerensen et Peter Bader. « Successful Haploidentical Stemcell Transplantation In a Patient with Fanconi Anemia and AML. » Blood 116, n^o 21 (19 novembre 2010) : 4586. http://dx.doi.org/10.1182/blood.v116.21.4586.4586.

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Abstract Abstract 4586 Allogeneic hematopoetic transplantation is the only curative treatment in patients with fanconi anemia indicated as soon transfusion dependency occurs and a matched family or unrelated donor is available. Overall survival for matched or nearly (5/6) matched unrelated donor transplantation improved since the introduction of fludarabine-based conditioning regimens from 30% to 53%. Transplantation after malignant transformation, in heavily pretransfused patients or from alternative donors is associated with a dismal outcome. We report the case of a 7 year old girl with fanconi anemia who developed AML-M4 (monosomy 7 and gain on chromosome 3) with 24% peripheral blasts during donor search for HCT. Remission was acchieved with a mild chemotherapeutic regimen consisting of a single dose of intravenous (300mg/m2) and intrathecal (40mg) cytarabine and daily oral thioguanine (cumulative dose: 930mg/m2). 4 weeks after diagnosis of AML, the girl was transferred for allogeneic PBSCT (CD3/19 depleted) from an unrelated mismatched (7/10) donor. Conditioning regimen consisted of Fludarabine, Busulfex, ATG and OKT-3. Unfortunately, mixed chimerism was observed and on day +21 graft rejection was diagnosed. The patient underwent a second PBSCT from the same donor after conditioning with Fludarabine, Alemtuzumab and Cyclophosphamide and 12 days later received a stem-cell-boost due to poor hematological regeneration. Prolonged aplasia under treatment with G-CSF was suspicious for non-engraftment. The indication for a third PBSCT with her haploidentical father as donor was set. Conditioning in the aplastic patient with elevated autologous T-Lymphocytes consisted of ATG only for 3 days. Full engraftment was achieved on day +11. CMV-reactivation was effectively treated with a combination therapy of valganciclovir and foscavir. One year after the third PBSCT, the patient is in continuing morphologic remission with normal Cytogenetics and complete donor chimerism. Mild Graft vs. Host Disease (Grade I, Skin) dissolved after short term therapy with Prednison. Still she suffers under minor restrictions in her every day life. We demonstrate here the case of a CMV-positive patient with fanconi anemia and AML, polytransfused (27 red-cell-packs and 94 platelet-transfusions) who survived mismatched PBSCT of her CMV-positive father after two failed PBSCT from an alternative 7/10-mismatched donor. To our knowledge this is the first patient with fanconi anemia and AML, successfully transplanted from an haploidentical donor. Disclosures: No relevant conflicts of interest to declare.

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Vrehen, H. M. « 1145 A nurse led out patient clinic for patients after stemcell transplantation ». European Journal of Cancer Supplements 1, n^o 5 (septembre 2003) : S348. http://dx.doi.org/10.1016/s1359-6349(03)91171-2.

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Sabo, Peter, Vahagn Makaryan, Tanoya Poulsen, Lital Povodovski, Yosef Dicken, Asael Herman, Rafi Emmanuel et David C. Dale. « CRISPR Mediated ELANE Single-Allele Knock-out Restores Proliferation and Myeloid Differentiation of Neutropenia Patient Derived BM HSCs ». Blood 136, Supplement 1 (5 novembre 2020) : 23. http://dx.doi.org/10.1182/blood-2020-137215.

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Background: Mutations in ELANE are a cause of cyclic and severe congenital neutropenia (SCN), predominantly autosomal dominant disorders. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease. Neutropenia occurs because mutant NE impairs survival and maturation of myeloid cells. More than 130 disease causing mutations in ELANE have been identified and cell permeable inhibitors of NE can correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). CRISPR/Cas9 knock-out (KO) of ELANE in patients' hematopoietic stem cells (HSCs) corrects myeloid cell differentiation, (Nasri et al Haematologica 2020:105:598), but this approach targets both alleles, eliminates normal NE and may impair innate immunity. Hypothesis: Selective, single allele editing is a preferred strategy for correcting ELANE associated neutropenia and other autosomal dominant disorders. Methods: Emendo Biotherapeutics developed CRISPR-associated allele specific KO based on single nucleotide polymorphisms (SNPs) located in the vicinity of ELANE. Three SNPs cover ~80% of the population. In the current study, editing of the mutated allele was achieved by targeting SNP rs1683564 located downstream to the 3'UTR of ELANE and mediating a biallelic break in intron 4, with excision of the 3'UTR, destabilized the mutated allele transcript. For editing we utilized RNPs assembled from Emendo's proprietary nuclease, OMNI-50, a guide targeting the SNP and a guide targeting a region in intron 4. OMNI-50 has low off-target activity and high allele specific editing when targeting either the reference or the alternative form of the SNPs. The efficacy of this composition was tested on normal and SCN patient-derived HSCs harboring the relevant SNP. (Figure 1) A patient's bone marrow CD34+ cells with the S126L mutation and rs1683564 were enriched using RosetteSep™ Progenitor Cell Enrichment Cocktail (Stemcell Technologies), purified by density gradient centrifugation using Lymphoprep (Stemcell Technologies). The purified CD34+ cells were expanded 4 days in StemSpan™ SFEM II media, supplemented with StemSpan™ CD34+ Expansion Supplement. Expanded CD34+ cells were further purified using StemSep™ Human CD34 Positive Cocktail and purity verified by FACS analysis using CD34 and CD45 antibodies. Purified CD34+ cells were cryopreserved using serum free CryoStor® CS10 media (Stemcell Technologies) and stored in Liquid Nitrogen Vapor Phase. Normal human blood CD34+ progenitor cells, harboring rs1683564 were similarly treated as the control. Allele specific excision was determined by ddPCR. To determine excision efficiency for the patient and control, we amplified two regions in ELANE, Exon 1 and Exon 5 (excised region), using two different probes labeled with FAM and HEX, respectively. We used OMNI-50 nuclease (Emendo Bio, Ness Ziona, Israel), electroporation (Lonza 4D nucleofector) and StemCell media (Stemcell Technologies, Vancouver, BC) to modify, grow and differentiate the CD34+ cells. We assessed myeloid cell proliferation and differentiation using daily cell counts, cytospins stained with Diff-Quik, and CD14, CD66b, CD15, CD16, CD11b labeling and flow cytometry. Results: Unedited patient cells demonstrated significant abnormalities in proliferation and differentiation. Cytospins from unedited patient CD34+ cells at 14 days showed block of myeloid differentiation and 4-fold greater monocytes compared to control, consistent with the hematopoietic defect in SCN patients. Single allele ELANE KO significantly improved these cellular abnormalities: total cell proliferation increased 41% and CD14/CD66b and CD15/CD11b positive cells increased by 107% and 65.5%, respectively. Studies are in progress for additional patients harboring different ELANE mutations. Conclusions: Single allele KO may resolve concerns about unwanted effects of total elimination of the normal gene products with gene editing for autosomal dominant diseases and be an effective strategy for treating ELANE associate neutropenia. Disclosures Sabo: Emendo BioTherapeutics: Research Funding. Makaryan:Emendo BioTherapeutics: Research Funding. Poulsen:Emendo BioTherapeutics: Research Funding. Povodovski:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dicken:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Herman:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Emmanuel:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dale:X4 Pharmaceuticals: Research Funding; Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Honoraria.

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Walcher, Lia, Nadja Hilger, Anja K. Wege, Franziska Lange, U. Sandy Tretbar, André‐René Blaudszun et Stephan Fricke. « Humanized mouse model : Hematopoietic stemcell transplantation and tracking using short tandem repeat technology ». Immunity, Inflammation and Disease 8, n^o 3 (11 juin 2020) : 363–70. http://dx.doi.org/10.1002/iid3.317.

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Ota, Akemi, Kazuaki Matsumura, Jun-Jae Lee, Shoichiro Sumi et Soung-Hyu Hyon. « StemCell Keep™ is Effective for Cryopreservation of Human Embryonic Stem Cells by Vitrification ». Cell Transplantation 26, n^o 5 (mai 2017) : 773–87. http://dx.doi.org/10.3727/096368916x692654.

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Safe and stable cryopreservation is critical for research involving human embryonic stem cells (hESCs). Dimethyl sulfoxide (DMSO) is a popular cryoprotective agent; however, its cytotoxicity cannot be ignored. Thus, there is a need for an alternate cryoprotectant. We reported previously that a novel cryopreservation reagent, StemCell Keep™ (SCK), was effective for cryopreserving human induced pluripotent stem cells (hiPSCs) by vitrification. Because hESCs and hiPSCs are not identical, the current study examined the use of SCK on hESCs. hESCs cryopreserved with SCK were thawed and cultured on SNL 76/7 cells, which were derived from a mouse fibroblast STO cell line transformed with neomycin resistance and murine LIF genes. After cryopreservation, cultured hESCs were assessed for their attachment ability and characterized by alkaline phosphatase (AP) and immunocytochemical (ICC) staining, fluorescence-activated cell sorting (FACS), reverse transcription polymerase chain reaction (RT-PCR), and karyotyping. The proliferation of SCK-cryopreserved hESCs cultured on SNL cells, or in feeder-free conditions, was higher than that of cells preserved in a solution of 2 M DMSO, 1 M acetamide, and 3 M propylene glycol (DAP). The cell number with SCK-cryopreserved hESCs was about twice that of hESCs cryopreserved in DAP. The pluripotency of SCK-cryopreserved hESCs was similar to that of DAP-cryopreserved hESCs based on AP staining. Data from ICC, FACS, and RT-PCR analyses showed that stem cell markers were continually expressed on SCK-cryopreserved hESCs. The teratoma assay showed that SCK-cryopreserved hESCs differentiated into three germ layers. Furthermore, SCK-cryopreserved hESCs had normal karyotypes. These data indicate that SCK was effective for cryopreservation of hESCs by vitrification.

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Apewokin, Senu, Elizabeth Coleman, Carol Enderlin, Julia Goodwin, Jeannette Lee, Stephen Erickson, Kent Mckelvey, Vinay Raj, Naveen Sanath Kumar et Zhou Daohong. « 438Genetic Variants Associated with the Development of Clostridium Difficile Infection during Autologous Stemcell Transplantation ». Open Forum Infectious Diseases 1, suppl_1 (2014) : S166—S167. http://dx.doi.org/10.1093/ofid/ofu052.304.

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Fliedner, M. « 1147 Non-adherence in hematology and stemcell transplant patients — is it worth worrying about ? » European Journal of Cancer Supplements 1, n^o 5 (septembre 2003) : S348. http://dx.doi.org/10.1016/s1359-6349(03)91173-6.

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Nooy, M. A., W. Fibbe, J. Ouwerkerk, D. van Benthem et E. M. Noordijk. « 1202 High-dose chemotherapy (HDC) with stemcell rescue for patients with metastatic Ewing's sarcoma ». European Journal of Cancer 31 (novembre 1995) : S251. http://dx.doi.org/10.1016/0959-8049(95)96448-m.

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Ota, Akemi, Suong-Hyu Hyon, Shoichiro Sumi et Kazuaki Matsumura. « Gene expression analysis of human induced pluripotent stem cells cryopreserved by vitrification using StemCell Keep ». Biochemistry and Biophysics Reports 28 (décembre 2021) : 101172. http://dx.doi.org/10.1016/j.bbrep.2021.101172.

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Versluys, Birgitta, M. Bierings, R. Schuurman, B. Ewijk et J. J. Boelens. « Clinical Impact of Respiratory Viruses in Haematopoietic Stemcell Transplantation ; a Firm Association with Bronchiolitis Obliterans. » Blood 106, n^o 11 (16 novembre 2005) : 3230. http://dx.doi.org/10.1182/blood.v106.11.3230.3230.

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Abstract In lung transplants respiratory viruses (RV) are associated with bronchiolitis obliterans (BO). Also in HSCT patients RV may progress to pneumonia and may trigger immunological mediated effects on lungfunction.We prospectively studied the clinical impact of RV in pediatric HSCT patients. Between 1/2004 and 7/2005 46 patient underwent 51 allo-HSCTs in our center. In all patients with respiratory symptoms viral PCRs (adeno-, human metapneumo-, influenza-, corona-, parainfluenza-, rhino- and RS-virus) were done on a nasopharyngeal washing, in addition to standard diagnostics. The severity and course of the respiratory symptoms was monitored. BO was defined as abnormalities in pulmonary function tests (> 20% decrease in FEV1) and/or on specific changes on HR-CT scan such as bronchial wall thickening, air trapping and mosaic parenchymal attenuation. 33 HLA-identical and 18 HLA-mismatched grafts were used from 11 sibs, 25 UDs, 12 cordbloods and 3 mismatched FDs. 8 grafts were T-cell depleted. Follow up was 9mths (range 1–18 mth). Overall survival is 67%. Acute-GvHD (> grade II) occured in 11/51 (21%) and cGVHD in 7/39 (18%; 4 extensive, including 3 BO, and 3 limited). Respiratory symptoms were seen in 25/51 HSCTs. In 6/25 patients a non-viral etiology was found: ARDS(2), VOD(2), IPS/DAH(2). The other 19 patients had viral disease; based on a positive PCR (16) or by clinical symptoms and exclusion of other causes (3). The onset of symptoms was within the 1st mth after HSCT (16), before HSCT (1) or after discharge (2). Rhinovirus (9), parainfluenza (3), influenza A (1) and multiple viruses (3; rhino/parainfluenza, adeno/parainfluenza, human metapneumo/rhino) were detected. 6/19 had upper respiratory tract infection (URTI) symptoms only, 11 were oxygen dependent, and 2 patients were ventilated. The patient with influenza-A was treated with a neuraminidase inhibitor. All others showed spontaneous recovery within 4–8 weeks. After resolution of symptoms and after tapering the immunosuppresion 3/19 patients developed BO (range: 3–8mth postHSCT). None of the other children in this cohort have developed BO. All BO patients underwent their first HSCT with a HLA-identical graft (2 sib, 1 UD). They were succesfully engrafted and had no signs of aGvHD. The severity of the initial respiratory symptoms, caused by rhinovirus (2) and para-influenza, was comparable to the other patients with RV infection. One patient died, the others are still receiving therapy. Respiratory problems are frequently seen in pediatric HSCT. RV were the most common cause in this cohort. The acute clinical course is usually mild, but in the longer term RV are firmly associated with BO. Our results might be an underestimate, due to short follow up of some patients. Monitoring viral infections with PCR of nasopharyngeal washing warrants early detection of a viral cause of the clinical syndrome. In such a case it might - paradoxically- be important to increase or prolong immunosuppressive treatment on a positive PCR only, or as soon as signs of BO develop.

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Suzliana, Rina Mutya, Tristiana Erawati, Cita Rosita Sigit Prakoeswa, Fedik Abdul Rantam et Widji Soeratri. « Pengaruh Asam Hialuronat-Space Peptide terhadap Karakteristik, Stabilitas Fisik Gel Amniotic Membran-Stemcell Metabolite Product ». JURNAL FARMASI DAN ILMU KEFARMASIAN INDONESIA 7, n^o 2 (30 novembre 2020) : 66. http://dx.doi.org/10.20473/jfiki.v7i22020.66-73.

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Pendahuluan: Amniotic membran stem cell metabolite product (AMSC-MP) merupakan metabolit produk yang diperoleh dari Amniotic membrane yang diisolasi dan dikultur yang kemudian ditumbuhkan dalam medium terkondisi. AMSC-MP mengandung banyak growth factor dan sitokin yang sangat berguna sebagai antiaging. Growth factor dan sitokin yang pada umumnya berukuran besar lebih dari 20 KDa menyebabkan AMSC-MP membutuhkan formulasi khusus untuk penggunaannya secara topikal. Pada penelitian ini digunakan asam hialuronat dikombinasi dengan SPACE peptide yang memiliki fungsi sebagai enhancer makromolekul untuk formulasi sediaan gel AMSC-MP sebagai antiaging. Untuk memastikan kualitas dan stabilitas suatu sediaan, maka perlu dilakukan uji karakteristik fisik dan uji stabilitas fisik sediaan tersebut. Tujuan: Mengevaluasi pengaruh penambahan asam hialuronat (0; 0,01; 0,02; 0,04 %) yang dikombinasi SPACE peptide terhadap karakteristik dan stabilitas dari formulasi sediaan gel AMSC-MP. Metode: Karakteristik fisik sediaan gel freeze dried AMSC-MP dengan penambahan asam hialuronat yang dikombinasi SPACE peptide dievaluasi dengan menggunakan parameter organoleptis (warna, bau, konsistensi dan tekstur), pengukuran pH dan kapasitas penyebaran. Pengujian stabilitas fisik dievaluasi dengan pengukuran pH dan kapasitas penyebaran sediaan setelah penyimpanan selama 28 hari. Hasil: Hasil uji karakteristik dan stabilitas fisik sediaan gel AMSC-MP menunjukkan penampilan fisik yang baik, gel transparan dengan konsistensi kental, lembut dengan bau yang khas. Nilai pH sekitar 5,26 - 5,37 dan kapasitas penyebaran pada 6,13 - 6,50. Semua formula stabil selama 28 hari penyimpanan. Kesimpulan: Penambahan kombinasi asam hialuronat dan SPACE peptide pada formulasi tidak merusak karakteristik dan stabilitas fisik sediaan gel AMSC-MP pada penyimpanan selama 28 hari.

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de Greef, Georgine E., Annemiek Broyl et Jaco Kraan. « The Mobilisation of Peripheral Blood Stemcells in Patients with High Risk or Relapsed Non-Hodgkin Lymphoma Does Not Lead to the Presence of Detectable Tumour Cells in the Stem Cell Harvest. » Blood 104, n^o 11 (16 novembre 2004) : 2873. http://dx.doi.org/10.1182/blood.v104.11.2873.2873.

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Abstract Consolidation treatment with high dose chemotherapy in combination with an autologous peripheral blood stem celll transplant. is widely used in patients with high risk non-Hodgkin lymphoma (NHL). Also this treatment has its place in patients with relapsed intermediate lymphoma. Earlier studies in patients with breast cancer or multiple myeloma showed evidence for (minimal) tumour contamination in the graft that might lead to the occurence of relapse. For patients with NHL this is less clear. For the present analysis 60 patients (pts) were included: 18 pts with primary high risk NHL, 6 patients with mantle cell lymphoma, and 36 pts with primary refractory or relapsed intermediate NHL. Patients with high risk NHL were treated with induction chemotherapy according to the Hovon-27 or Hovon-40 protocol(www.hovon.nl). Two patients (1 pt with T-Lymphoblastair lymphoma, 1 pt with Burkitt Lymphoma ) received intensive induction chemotherapy according to our leukemia protocols. Stem cells were collected as early as possible, provided the peripheral blood and bone marrow showed no histological evidence of NHL and < 20% lymfocytes. In the patients with mantle cell lymphoma stem cells were collected during the induction chemotherapy (Hovon-45 protocol:www.hovon.nl), irrespective of their presence in the bone marrow. The patients with primary refractory or relapsed intermediate NHL received reinduction chemotherapy with DHAP (Dexamethason, Cytarabin, Cisplatin) or EMP (Etopside, Mitoxantrone, Prednisone). After two or three courses peripheral blood stem cells were mobilised with G-CSF (Neupogen, Amgen) 10 μg/kg sc daily starting day 5 and harvested for autologous stem cell transplant. Before the start of mobilisation patients were required to be responsive to the induction chemotherapy and to have a bone marrow without histological and immunological detectable tumour cells. In all patients a sample of the first day stem cell collection was analyzed with the use of three or four colour Flowcytometry. The sensitivity of the assay was 1/ 104–105 lymphoma cells. Only in 3 pts tumour cells were found at a level > 0.05 %. Interestingly two patients had a primary diagnosis of diffuse large B-cell lymphoma T-cell/ histiocyte rich variant. Both pts relapsed: one within 6 months and the other one within 12 months.The third patient suffered from a anaplastic large cell T-cell lymphoma and relapsed after 15 months. In all other patients no lymphoma cells were detectable in the stemcell harvest at a minimum level of <0.02%. Follow up showed a relapse within 12 months in 16 of 36 patients with relapse or primary refractory NHL (44%) and 6 of 24 patients with high risk NHL (25%). In conclusion, in the vast majority of the above patients, no circulating lymphoma cells were detectable with the use of flowcytometry in the stemcell harvest. Therefore we conclude that with the present techniques and sensitivity immunological analysis of the graft does not give additional information and therefore is not necessary in this patient group. Provided the bone marrow before collection is free of lymphoma cells, we feel that the currently used mobilisation regimen is safe in these patients and does not lead to the presence of detectable tumour cells in the collected stemcells.

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Douek, Daniel C., Robert A. Vescio, Michael R. Betts, Jason M. Brenchley, Brenna J. Hill, Lan Zhang, James R. Berenson, Robert H. Collins et Richard A. Koup. « Assessment of thymic output in adults after haematopoietic stemcell transplantation and prediction of T-cell reconstitution ». Lancet 355, n^o 9218 (mai 2000) : 1875–81. http://dx.doi.org/10.1016/s0140-6736(00)02293-5.

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Su, Z., J. Wu, N. Kirby, Z. Li et I. Barani. « SU-E-T-568 : Hippocampus and Neural Stemcell Sparing Using Proton Therapy in Whole Brain Irradiation ». Medical Physics 39, n^o 6Part19 (juin 2012) : 3836. http://dx.doi.org/10.1118/1.4735657.

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Jurczak, Wojciech, Marta Szostek, Zbigniew Rudzki, Dorota Krochmalczyk, Joanna Wegrzyn, Jaroslaw Czyz et Aleksander B. Skotnicki. « Impaired Clonogenic Capacity Contributes to Bone Marrow Hypoplasia and Late Hematological Recovery after Zevalin in the Low-Grade NHL Patients. » Blood 104, n^o 11 (16 novembre 2004) : 4610. http://dx.doi.org/10.1182/blood.v104.11.4610.4610.

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Abstract Chemoimmunotherapy with Zevalin (anty CD20,90Y) is a new therapeutic approach in the resistant or relapsed low grade malignant lymphoma patients. The most important side effect observed in the up to date studies is related to transient peripheral blood cytopenias. In an attempt to evaluate the involved pathogenetic mechanisms of the above findings, the blood and bone marrow samples from 8 low grade NHL patients were prospectively tested for the peripheral blood count, the clonogenic capacity and the histopathology abnormalities (trephine biopsies) before and every 14 days after Zevalin administration until recovery. In the majority evaluated patients the mild thrombocytopenia (30-55000/μl) was observed 4 weeks after Zevalin therapy. Bone marrow mononuclear cells were cultured in methylcellulose assay using combination of recombinant CSF (IL-3, IL-6, stem cell factor, erythropoietin; Methocult, StemCell, Canada), at a concentration of 1x10^5 cells, in 35 mm gridded Petri dish and incubated at 37°C, in a humidified atmosphere of 5% CO2. The hematopoietic colonies (CFU-GM, BFU-E and CFU-GEMM) were evaluated on 14th d. of the culture. The in vitro CFU-Meg formation in the serum-free collagen-based semisolid culture (Megacult, StemCell, Canada) were also studied, stained after 12 days with a monoclonal antibody against CD 41. In the results of our studies the significant inhibition of colony formation was found already in 7th day for CFU-GM and BFU-E and starting from 14th day after Zevalin administration for CFU-Meg. In the subsequent cultures evaluated during 4–8 weeks after Zevalin therapy the three lineages hematopoietic clonogenic capacity returned to the pre-radiotherapy values. In the repeated trephine biopsis several morphological changes were observed: oedema of stromal cells, the relative increase of erythropoietic components with inverted proportion of granulo- to erythropoiesis. In conclusion, chemoimmunotherapy with Zevalin resulted in the reversible peripheral cytopenia parallely with transient damage to bone marrow hematopoiesis and in vitro clonogenic capacity.

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Leitner, G. C., A. Bartuschka et A. Schulenburg. « Measurement of Aldehyde Dehydrogenase Activity in Allogeneic or Autologous Peripheral Stemcell Grafts May Substitute Time Consuming Cells Cultures ». Biology of Blood and Marrow Transplantation 17, n^o 2 (février 2011) : S206. http://dx.doi.org/10.1016/j.bbmt.2010.12.165.

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Shamim, Zaiba, Stephanie Thiant, Sylvie Faucher, Bachra Choufi, Lars Ryder, Ibrahim Yakoubagha et Klaus Muller. « Plasma Levels of Soluble Interleukin-7 Receptor Alpha (sIL-7Ra) and IL-7Ra Polymorphism After Allogeneic Stemcell Transplantation »,. Blood 118, n^o 21 (18 novembre 2011) : 4012. http://dx.doi.org/10.1182/blood.v118.21.4012.4012.

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Abstract Abstract 4012 Background: Interleukin-7 (IL-7) is a cytokine essential for T cell development in the thymus and maintenance of peripheral T cells. IL-7 binds to cellular IL-7 receptors (IL-7Ra-common g chain heterodimer), in competition with a soluble form of the receptor, shed by the cells (sIL-7Ra). We have identified single nucleotide polymorphisms in the exons of the gene encoding IL-7Ra (+510T/C rs1494558, +1237 G/C rs1494555, 2087 C/T rs6897932), and previous results by us and by others indicate that IL-7R SNPs are associated with aGvHD and mortality after SCT. Moreover, the biological significance of +2087 C/T SNP has been suggested by the finding of elevated serum levels of sIL-7Ra in healthy individuals with 2087CC (Haplotype 4), also associated with increased alternative splicing of exon 6 and a higher frequency of recent thymic emigrants. We hypothesized that sIL-7Ra levels during SCT are influenced by genetic polymorphism and may play a role in immune reconstitution after SCT. Aims: 1) To investigate sIL-7Ra levels during SCT along with IL-7Ra genotypes and 2) to evaluate associations between sIL-7Ra levels and immune reconstitution and outcome in SCT. Patients and Methods: 122 patients undergoing SCT for haematological malignancies after either myeloablative (n= 52) or reduced intensity conditioning (n=70) were included. Donors were either matched siblings (n=68) or matched unrelated donors (n=54), and the patient age at the time of transplant was 50 years (6–67) (median (range)). sIL-7Ra levels were measured in plasma by a quantitative bead capture assay. IL-7Ra SNPs were determined by a SSP-PCR system and IL-7 was tested by ELISA (R&D) (n=77). Results: sIL-7Ra levels decreased during the course of transplantation from 113 ng/ml (32–558) at day −15 to 48 ng/ml (32–195) at day +14 (p=0.0001), and reached baseline levels again at day +60. sIL-7Ra levels were not associated with the intensity of the conditioning regimen. This pattern appeared to be inversely mirrored by the IL-7 levels, which increased from baseline values at day –14 of 2.4 pg/ml (0.3–17.6) to 11.3 pg/ml (2.0–30.2) (p<0.0001) at day +14, followed by a gradual decline down to baseline values at day +60. sIL-7Ra levels at day +90 were reduced in patients transplanted with donors carrying IL-7Ra 2087T allele, in line with our previous findings in healthy individuals (105 ng/ml (42–274) vs. 152 ng/ml (20–971), p=0.0015). In addition, post-transplant sIL-7Ra levels correlated with pre-transplant sIL-7Ra levels (r=0.39 p=0.0032), indicating that patient related factors in addition to the genotype of the donor lymphocytes may play a role in the regulation of sIL-7Ra levels post-transplant. sIL-7Ra appeared to be predictive of the rate of immune reconstitution by the finding that sIL-7Ra at day +14 correlated significantly with total lymphocyte counts post-transplant (day +90: r=0.28 (p=0.031) and day +180: r=0.55 (p<0.0001)). In contrast, IL-7Ra genotypes were not associated with immune lymphocyte counts post-transplant and early post-transplant IL-7 levels did not correlate significantly with lymphocyte counts at any stage. Outcomes: There was a trend towards an association between high sIL-7Ra levels and increased overall survival (p=0.057), but sIL-7Ra levels were unrelated to the occurrence of aGvHD. However, the IL-7/sIL-7Ra ratio at day +14 was significantly higher in patients with grade 2–4 aGvHD compared to grade 0–1 aGvHD (0.29 (0.04–0.73) vs 0.19 (0.01–0.8), p=0.033). Conclusion: The data of the present study indicates that sIL-7Ra levels after SCT are determined by the IL-7R 2087 genotype of the donor in addition to patient related factors. sIL-7Ra levels in the early phase post transplant is associated with the rate of lymphocyte recovery post-SCT. Thereby this study adds to the growing evidence suggesting the importance of the IL-7 axis in SCT. The study further suggests that monitoring sIL-7Ra levels post-transplant may help to guide the clinical use of IL-7. Disclosures: No relevant conflicts of interest to declare.

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Hussain, Ali, Moazzam Shahzad, Sakina Abbas, Junaid Khan, Ali Hassan Mushtaq, Muhammad Fareed Khalild, Mohammad Ammad Ud Din et al. « Outcomes with Allogeneic Hematopoietic Stemcell Transplantation in NPM1-Mutated Acute Myeloid Leukemia : A Systematic Review And Meta-Analysis ». Transplantation and Cellular Therapy 29, n^o 2 (février 2023) : S125—S126. http://dx.doi.org/10.1016/s2666-6367(23)00224-5.

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Bonfichi, Maurizio, Ercole Brusamolino, Carmela Marseglia, Chiara Rusconi et Mario Lazzarino. « Evaluation of Progenitor Cells (CFU-GM, CFU-GEMM, BFU-E, CD34+ Cells) and White Blood Cells in Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma Treated with Rituximab-CHOP-14 and Supported with Pegfilgrastim. » Blood 106, n^o 11 (16 novembre 2005) : 4245. http://dx.doi.org/10.1182/blood.v106.11.4245.4245.

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Abstract Purpose: To investigate the behavior of CFU-GM, CFU-GEMM, BFU-E, CD34+ cells and white blood cells before and during six courses of dose-dense immune-chemotherapy (R-CHOP-14) supported by pegfilgrastim in eight patients affected with newly diagnosed diffuse large B cell lymphoma (3 men, 5 women, aged 18–51). Patients and Methods: The standard-dose CHOP chemotherapy was delivered every 14 days, preceded on day 1 by rituximab (375 mg/m2) and followed on day 3 by pegfilgrastim (6 mg per cycle). In each course of therapy, the evaluation was performed before treatment (day 1) and on days +3, +6, +8, +10 and +13. Hemopoietic progenitors were assayed in a complete StemCell medium (MethoCultTM GF H4434 StemCell, Canada) and counted with an inverted microscope after 14-day incubation at 37°C, in 5% CO2. CD34+ cells were evaluated with flow cytometry (EPICS XL, Beckman Coulter); WBC were evaluated with an automatic counter (ADVIA 120 Bayer). Results: The peak of CFU-GM, CFU-GEMM, BFU-E colonies and of CD34+ cells was invariably reached on day +13 and the highest absolute values were detected after the first cycle of therapy: CFU-GM/ml 3704±738; CFU-GEMM/ml 199±62; BFU-E/ml 8848±3499; CD34+cells/μL 33±3. In the subsequent courses, the number of both hematopoietic colonies and of CD34+ cells progressively lowered; however, the median value of CD34+ cells was still over 20/μL (range 20–35/μL) before the start of the fourth cycle of R-CHOP-14. A significant direct correlation was found between the number of CFU-GM, CFU-GEMM and BFU-E colonies and that of CD34+ cells (p<0.05). The WBC peak was constantly reached on day +6, with a mean value, all cycles considered, of 32±14 x 109/l. Comments: Hemopoietic progenitor cells kynetic showed that colonies and CD34+ cells started to increase at the WBC nadir. Most important, the level of circulating CD34+ cells at day 13 of the first three cycles of therapy was above 20/μL which we consider permissive for a productive leukaferesis; this observation adds further evidence of the pegfilgrastim progenitor cells mobilizing capacity.

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Janicsák, Henrietta, Tamás Masszi, Péter Reményi, Gabor S. Ungvari et Gábor Gazdag. « Impact of the type of hematopoietic stem-cell transplant on quality of life and psychopathology ». Ideggyógyászati szemle 76, n^o 1-2 (2023) : 25–35. http://dx.doi.org/10.18071/isz.76.0025.

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Background and purpose – Despite the decrease in transplant-related mortality, patients who receive hematopoietic stemcell transplants often suffer from short-and long-term morbidities, poorer quality of life, and psychosocial functioning deficits. Several studies have compared the quality of life and affective symptoms of patients after undergoing autologous and allogeneic hematopoietic stem-cell transplants. Some studies have reported similar or greater quality of life impairments in allogeneic hematopoietic stemcell recipients, but the findings have been inconsistent. Our purpose was to examine the influence of the type of hematopoietic stem-cell transplantation on the quality of life and affective symptoms of patients. Methods – The study sample comprised 121 patients with various hematological diseases who underwent hematopoietic stem-cell transplantation at St. István and St. László Hospitals, Budapest. The study had a cross-sectional design. Quality of life was evaluated using the Hungarian version of the Functional Assessment of Cancer Therapy– Bone Marrow Transplant scale (FACT-BMT). Anxiety and depressive symptoms were assessed using Spielberger’s State and Trait Anxiety Inventory (STAI) and the Beck Depression Inventory (BDI), respectively. Basic sociodemographic and clinical variables were also recorded. Comparisons between autologous and allogeneic recipients were analyzed using a t-test when the variables were normally distributed and a Mann–Whitney U test otherwise. A stepwise multiple linear regression analysis was performed to identify the risk factors that contributed to the quality of life and the affective symptoms in each group. Results – Quality of life (p=0.83) and affective symptoms (pBDI=0.24; pSSTAI=0.63) were similar between the autologous and allogeneic transplant groups. The BDI scores of allogeneic transplant patients indicated mild depression, but their STAI scores were similar to those of the general population. Allogeneic transplant patients with symptoms of graft-versus-host disease (GVHD) experienced more severe clinical conditions (p=0.01), poorer functional status (p<0.01) and received more immunosuppressive treatment (p<0.01) than those without graft versus host disease. Patients suffering from graft versus host disease experienced more severe depression (p=0.01), and constant anxiety (p=0.03) than those without graft versus host disease. Quality of life was affected by depressive and anxiety symptoms and psychiatric comorbidity in both the allogeneic and autologous groups. Conclusion – Graft versus host disease-related severe somatic complaints seemed to influence the allogeneic transplant patients’ quality of life by inducing depressive and anxiety symptoms.

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Latham, Stephen R. « Between Public Opinion and Public Policy : Human Embryonic Stem-Cell Research and Path-Dependency ». Journal of Law, Medicine & ; Ethics 37, n^o 4 (2009) : 800–806. http://dx.doi.org/10.1111/j.1748-720x.2009.00451.x.

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My aim in this paper is simply to show that, in bioethics no less than in other areas of health care, policy in democracies is shaped not only by principles and values, but also — and to some extent independently — by the shape and history of particular political institutions and past policies. “Path dependency,” or what one scholar has called the “accidental logics” of already-existing institutions, condition and guide national policy choices. These institutional and historical pressures can even create substantial policy divergences between quite likeminded nations. I shall illustrate the point using some comparative data about national policies regarding research on human embryonic stem cells. The fact that gaps can develop between values and policies is readily visible to anyone who compares national stemcell research policies to the expressed attitudes of the citizens of various democratic countries regarding human embryonic stem-cell research. The role of path dependency and the accidental logics of institutional structure in creating those gaps can be illustrated by tracking down the details of the development of human embryonic stem cell policies in a few different countries.

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Kröger, Nicolaus. « Prevention of Chronic Graft-versus-host Disease and the Unique Role of Anti-human T-lymphocyte Immune Globulin ». European Oncology & ; Haematology 12, n^o 02 (2016) : 93. http://dx.doi.org/10.17925/eoh.2016.12.02.93.

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Chronic graft-versus-host disease (GVHD) is a major cause of late morbidity and mortality post-allogeneic haematopoietic stemcell transplantation. Compared with acute GVHD, for which progress in preventative measures have been made, chronic GVHD describes a more diverse syndrome, and may adversely affect almost all organs in the body. A new prospective, multicentre, open-label, randomised phase III study (n=168) showed that the use of anti-human T-lymphocyte immune globulin (ATLG) in a myeloablative conditioning regimen for patients with acute leukaemia led to a significantly lower rate of chronic GVHD post-allogeneic transplantation compared with those receiving the same regimen without ATLG. Importantly, no increased rate of relapses in the patients who received ATLG was seen compared with those who did not. Thus, there was no apparent impairment in the graft-versus-leukaemia effect in ATLG-treated patients. The study was terminated at 2 years and more evidence about the long-term effect of ATLG on survival and GVHD relapses beyond this time-point are needed. Nonetheless, the findings represent a significant advance in the prevention of chronic GVHD.

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Wermke, M., J. Eckoldt, K. S. Götze, S. A. Klein, L. C. de Wreede, G. Bug, F. Stölzel et al. « Labile Plasma Iron Predicts for Survival in Patients Undergoing Allogeneic Stemcell-Transplantation – Results from the Prospective Multicenter German-Austrian Allive Trial ». Leukemia Research 55 (avril 2017) : S5—S6. http://dx.doi.org/10.1016/s0145-2126(17)30124-8.

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Inoue, Kana, Akiko Sumitomo, Natsumi Hasegawa, Ayuko Kasai, Kenji Yonezawa, Norinaga Urahama et Mitsuhiro Ito. « Involvement of Transcriptional Mediator Subunit TRAP220/MED1 in Action of Niche Cells to Support Hematopoietic Stem/Progenitor Cells ». Blood 112, n^o 11 (16 novembre 2008) : 3581. http://dx.doi.org/10.1182/blood.v112.11.3581.3581.

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Abstract The mammalian TRAP/Mediator complex is a master transcriptional regulatory complex that integrates signals of diverse activators and recruits RNA polymerase II and other general factors to activate transcription. The TRAP220/MED1 subunit was originally identified as a ligand-dependent coactivator specific for nuclear receptors. We have previously shown through biochemical and mouse genetic studies that MED1 is essential for embryogenesis, cell growth/differentiation and homeostasis, and that it stimulates nuclear receptor-mediated myelomonopoiesis. MED1 also integrates other activators such as GATA-1 and C/EBPβ and appears to mediate erythropoiesis as well. The niche cells in the bone marrow plays a pivotal role in the maintenance of hematopoietic stem/progenitor cells (HSPCs). In this study, we employed mouse embryonic fibroblasts (MEFs) as a model to analyze the role of MED1 in the niche, since MEFs have a mesenchymal feature with the osteoblastic precursor lineage and are known to support HSPCs. To establish an experimental system, we crossed Med1 and p53 double knockouts to obtain Med1+/+/p53−/− and Med1−/−/p53−/− E10.0 embryos from a single female and prepared stable MEF lines. Then the Med1−/−/p53−/− MEFs were stably transfected with a MED1 expression vector (Rev-Med1−/− MEFs) or a control empty vector. When normal mouse bone marrow cells were cocultured with these MEFs treated with mitomycin C for a short period of 2 weeks, cell counts, live cells (MTT assay) and a DNA synthesis (BrdU incorporation) of marrow cells were measured. The number of live cells as well as DNA synthesis on Med1−/− MEFs was significantly decreased during this period, but those on Rev-Med1−/− MEFs recovered to the control levels. Thus the growth stress on MEFs appears to be attenuated on Med1−/− MEFs. When apoptosis of the marrow cells was measured, both the FITC-dUTP incorporation by TdT and annexin V/PI double positive cells were lower for Med1−/− MEFs, indicating that apoptosis was also attenuated. We next assessed the role of MED1 in MEFs to support long-term bone marrow culture. After bone marrow cells were cultured on mitomycin C-treated MEFs for 8 weeks in Myelocult M5300 (StemCell Technologies) or IMDM supplemented with BIT9500 (StemCell Technologies) and LDL, progenitor cells (adherent and nonadherent) were collected and cultured in complete methylcellulose (Methocult M3434; StemCell Technologies), and colonies were counted. The number of both myeloid and erythroid colonies were significantly attenuated (0 to 40% depending on experimental conditions) for cells on Med1−/− MEFs, but colonies for cells cultured on Rev-Med1−/− MEFs recovered to the control level. In order to exclude the possibility that lot differences among MEFs or p53 depletion might have affected the results, we next prepared primary Med1+/+ and Med1−/− MEFs by crossing Med1+/− mice and conducted the long-term culture experiments using these MEFs. The attenuated number of colonies for cells cocultured with Med1−/− MEFs (circa 10% of the control) was reproduced repeatedly, indicating that the observed role of MED1 in MEFs to support HSPCs is intrinsic. Since MED1 converges signals from a series of activators on specific promoters and activates transcription, one or some products of the downstream target genes in MEFs may be responsible for the observed activity to maintain HSPCs. In search for candidate MED1 target gene products among a series of known molecules that possess an activity on HSPCs, only the expression of osteopontin was found to be attenuated in Med1−/− MEFs and reverted in Rev-Med1−/− MEFs. Other factors including Angiopoietin-1 and Jagged-1 were comparable. This fact contrasts with the previous observation of osteopontin knockouts where the null niche cells that restricted the size of HSPC number overexpressed these factors. We next assessed the role of MED1 on the osteopontin promoter. We focused on vitamin D receptor (VDR) and Runx2 among the activators and tested MEFs by luciferase reporter assays. The basal level of transcription without any activators in Med1−/− MEFs was about half of the control. Moreover, both the activation by Runx2 and the liganddependent VDR function were significantly attenuated in Med1−/− MEFs. These results indicate that transcriptional coactivator MED1 in niche cells plays an important role in HSPCs support, and that osteopontin may be one of the downstream candidate target genes for MED1.

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Ampudia, Jeanette, Dalena Chu, Jana Badrani, Taylor Doherty, Stephen Connelly et Cherie Tracy Ng. « Impact of Itolizumab on the Expression of CD6 and the Function of Effector T Cells ». Blood 136, Supplement 1 (5 novembre 2020) : 25–26. http://dx.doi.org/10.1182/blood-2020-141638.

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Introduction: CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory (AI) diseases. In GVHD, CD6 is expressed on reconstituting T cells soon after transplant (Rambaldi et al., 2019), including Th1 and Th17 cells which are both implicated in the induction and pathogenesis of acute GVHD (aGVHD). CD6 is highly expressed on these cells and promotes immune synapse formation, T-cell activation, and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). Furthermore, studies have demonstrated that ex-vivo depletion of CD6+ donor cells prior to hematopoietic cell transplantation (HCT) decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998), highlighting the importance of CD6. While the contribution of CD6 to T cell activation has been well described, less is known regarding the expression levels and role of CD6 on effector and memory T cells (Teff) which are prominent in all diseases including aGVHD. Consequently, the aim of this study was to determine the role of CD6 specifically on effector T cells, and further illuminate the mechanism of itolizumab, an anti-CD6 monoclonal antibody. Methods: Naïve T cells were enriched from frozen PBMCs via a naïve T cell magnetic separation kit (Stemcell). Naïve T cells were polarized towards a Th1 phenotype for 6 days with a Th1 differentiation cocktail (Stemcell) and CD3/CD28 T cell activator (Stemcell) and rested overnight prior to entering restimulation conditions. Naïve T cells were polarized towards a Th17 phenotype for 8 days with IL-6, IL-1β, TGF-β, IL-23, anti-IL-4 and IFN-γ, the CD3/CD28 T cell activator was used to activate the T cells. To re-stimulate differentiated T cells, anti-CD3 mAb and ALCAM-Fc or anti-CD3 mAb alone were coated on 48-well plates overnight at 4oC. Th1 or Th17 T cells were labeled with CFSE and seeded with isotype control or itolizumab for 72hrs. Cells were collected for flow cytometry analysis and supernatant collected for relevant cytokine detection. To assess surface levels of CD6, cryopreserved PBMCs were thawed and incubated with itolizumab or isotype at 37oC for specific timepoints. Following incubation, cells were washed and stored at 4oC for subsequent staining. All samples were stained at the same time and surface levels of CD6 was detected using a monoclonal anti-CD6 antibody that does not compete with itolizumab. Results: Blockade of the CD6 pathway, using itolizumab during restimulation of differentiated Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in cell size. An average of a 40% decrease in CFSE proliferation was observed across multiple donors. Furthermore, treatment of Teff cells with itolizumab resulted in a significant decrease in expression level of T cell markers of activation and exhaustion such as CD25, PD-1 and Tim3. This effect was exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. When levels of CD6 were assessed, CD45RO+ (Teff/mem) cells expressed higher levels than CD45RA+CD45RO- (Tnaive). Itolizumab treatment of in vitro generated CD45RO+ T cells inhibited this stimulation-induced increase in CD6 in a dose-dependent manner, suggesting that the drug may modulate surface CD6 expression as a mechanism separate from physical blockade of CD6. Conclusions: These findings are the first to characterize the CD6-ALCAM pathway as a key regulator of differentiated effector T-cell function. Modulation of activation markers and CD6 itself by itolizumab, suggest modulation of Teff activity by both direct and indirect inhibition of CD6 signaling. These data further support targeting the CD6-ALCAM pathway to inhibit both naïve and effector T cell populations in aGVHD. Disclosures Ampudia: Equillium: Current Employment, Current equity holder in publicly-traded company. Chu:Equillium: Current Employment, Current equity holder in publicly-traded company. Doherty:Equillium Inc.: Research Funding. Connelly:Equillium: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Ng:Equillium: Current Employment, Current equity holder in publicly-traded company.

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Yang, Tingting, Xueping Luo, Qing Yang, Hongchao Chen, Yi Luo, Yanmin Zhao, Xiaoyu Lai et al. « The Effect of Continuous Screening of Carbapenem-Resistant Enterobacteriaceae on Theprevention and Control of Bloodstream Infections in Patients with Hematopoietic Stemcell Transplantation ». Blood 134, Supplement_1 (13 novembre 2019) : 5606. http://dx.doi.org/10.1182/blood-2019-127599.

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Carbapenem-resistant Enterobacteriaceae (CRE) bloodstream infections (BSIs) have become emerging cause of death in patients with hematopoietic stem cell transplantation (HSCT). Patients underwent HSCT from September 2017 to June 2018, and from July 2018 to February 2019, were assigned as single screening group and continuous screening group; patients transplanted from January 2016 to August 2017 were assigned as control group. Continuous screening significantly improved the CRE gut detection rate compared with single screening (10% vs 1.5%, p=0.001). The CRE infection rate in the pre-intervention period, single screening period and continuous screening period were 1.6%, 2% and 0, respectively; while related mortality was were 66.7%, 50% and 0, respectively. The time from the onset of symptoms of infection to use of tigecycline in survival patients with BSIs were shorter than died patients (24 hours vs 72 hours). For 11 CRE carriers with neutropenic fever, all received tigecycline therapy-based therapy (time from detection to therapy, -3 to 17 days) and did not develop BSIs. These results suggest that continuous screening can more effectively identify high-risk groups of CRE colonization and guide targeted preemptive treatment in the presence of infection symptoms, so as to reduce the incidence of BSIs and improve the prognosis of patients. Disclosures No relevant conflicts of interest to declare.

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Simbulan-Rosenthal, Dougherty, Vakili, Ferraro, Kuo, Alobaidi, Aljehane et al. « CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis ». Cancers 11, n^o 10 (3 octobre 2019) : 1490. http://dx.doi.org/10.3390/cancers11101490.

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CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably acancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+)‘melanoma-initiating cells’ are associated with chemoresistance, contributing to poor patientoutcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magneticactivatedcell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealedthat CD133(+) cells are significantly more invasive than CD133(−) cells. Conditional reprogrammingof BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stemcell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cellsshowed upregulated CD133 expression, and consequently were more invasive and metastatic thanBAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAKR-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAKR-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA orCRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels,doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9concentrations. CD133 may therefore play an essential role in invasion and metastasis viaupregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target forintervention in melanoma.

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Palumbo, Paola, Francesca Lombardi, Giuseppe Siragusa, Soheila Dehcordi, Sabino Luzzi, AnnaMaria Cimini, Maria Cifone et Benedetta Cinque. « Involvement of NOS2 Activity on Human Glioma Cell Growth, Clonogenic Potential, and Neurosphere Generation ». International Journal of Molecular Sciences 19, n^o 9 (17 septembre 2018) : 2801. http://dx.doi.org/10.3390/ijms19092801.

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Aberrant nitric oxide synthase 2 (NOS2) expression has been suggested as an interesting therapeutic target that is being implicated as a component of the molecular profile of several human malignant tumors, including glioblastoma, which is the most aggressive brain tumor with limited therapeutic options and poor prognosis. The aim of the present work was to evaluate the effect of 1400W, a specific NOS2 inhibitor, on human glioma cells in terms of clonogenic potential, proliferation, migration rate, and neurosphere generation ability. NOS2 expression was determined by Western blotting. Nitric oxide (NO) production was measured through nitrite level determination. The trypan blue exclusion test and the plate colony formation assay were performed to evaluate cell proliferation and clonogenic potential. Cell proliferation and migration ability was assessed by the in vitro wound-healing assay. Neurosphere generation in a specific stemcell medium was investigated. NOS2 was confirmed to be expressed in both the glioma cell line and a human glioma primary culture, and overexpressed in relative derived neurospheres. Experiments that aimed to evaluate the influence of 1400W on U-87 MG, T98G (glioblastoma cell lines) and primary glioma cells sustained the crucial role played by NOS2 in proliferation, colony formation, migration, and neurosphere generation, thus supporting the emerging relevance of a NOS2/NO system as a prognostic factor for glioma malignancy and recurrence.

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Kokaji, Andy, Neil MacDonald, Maureen Fairhurst et Terry Thomas. « A rapid and efficient method for the isolation of distinct human regulatory T cell populations using combinations of antibody mediated buoyant density centrifugation and/or column-free immunomagnetic cell separation (143.41) ». Journal of Immunology 184, n^o 1_Supplement (1 avril 2010) : 143.41. http://dx.doi.org/10.4049/jimmunol.184.supp.143.41.

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Abstract Regulatory T cells (Tregs) are a specialized subset of T cells that play a key role in immune regulation. Harnessing the suppressive function of Tregs is a major area of interest as they hold great potential for the treatment of autoimmune disorders. The first step towards quality Treg research is the isolation of highly purified, functional Tregs. With unique cell isolation platforms, STEMCELL has developed a full range of products for the rapid and efficient isolation of highly functional Tregs from virtually any peripheral blood sample. RosetteSep is an antibody mediated buoyant density centrifugation method used to isolate unlabelled cells specifically from whole blood or buffy coat samples. EasySep is an immunomagnetic cell separation method used to isolate cells from fresh or previously frozen PBMCs. Treg pre-enrichment is achieved by antibody mediated crosslinking of unwanted cells to either red blood cells (RosetteSep) or magnetic particles (EasySep) allowing their removal by Ficoll centrifugation or magnetic separation, respectively. RosetteSep or EasySep pre-enriched Treg populations consist of CD4+, CD4+CD127low or CD4+CD127lowCD49d- T cells. Pre-enriched Tregs can be further purified using EasySep positive selection to isolate Tregs expressing high levels of cell surface CD25. Purities of 85% +/- 10% CD4+CD25highFOXP3+ human Tregs can be achieved depending on the Treg population. From start to finish, Treg isolations can be completed in less than 3 hours.

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Dean, Melinda M., Thu V. Tran, Yu Ji, Tanya Powley, Yuan Tong Gu, Emilie Sauret et Robert L. Flower. « Development of a Semi-automated Monocyte Monolayer Assay (MMA) to Determine the Clinical Significance of Red Cell Alloantibodies ». Journal of Immunology 204, n^o 1_Supplement (1 mai 2020) : 86.55. http://dx.doi.org/10.4049/jimmunol.204.supp.86.55.

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Abstract BACKGROUND One risk of allogeneic blood transfusion is the development of alloantibodies against red blood cell (RBC) antigens not found on the recipient’s RBC. The monocyte monolayer assay (MMA) is an in vitro model which has been used to determine the clinical significance of alloantibodies and predict post-transfusion RBC survival. We aimed to develop a semi-automated procedure to reduce assay time and facilitate improved patient management. METHODS PBMCs isolated using Sepmate tubes (STEMCELL Technologies) were added to Lab-Tek 8-well chamber slides (37°C, 1 hr, CO2). Antigen matched RBC were sensitised with plasma with known anti-RBC antibodies (anti-D n=3, anti-Yta n=4) (37°C, 1 hr, CO2). A modified Wright-Giemsa staining protocol was performed (Aerospray Hematology Pro 2 stainer, ELITechGroup) and the proportion of bound and/or phagocytosed RBC was determined via microscopy (>5% considered clinically significant). RESULTS The introduction of PBMC isolation using sepmate tubes and automated cell staining reduced technical requirements, improved assay reproducibility and reduced assay time by >2 hours. For the alloantibodies tested, 67% of the anti-D and none of the anti-Yta were clinically significant. CONCLUSIONS We developed a MMA with improved reproducibility and reduced turnaround time. This assay will improve blood transfusion safety and facilitate better patient management by reducing the risk of incompatible blood transfusion.

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Lange, Andrzej, Dorota Dlubek, Barbara Wysoczanska, Daria Drabczak-Skrzypek et Emilia Jaskula. « An Evidence That Mesenchymal Stem Cells Are Not Replaced by Peripheral Blood Stem Cell Allografts in CML Patients. » Blood 106, n^o 11 (16 novembre 2005) : 4875. http://dx.doi.org/10.1182/blood.v106.11.4875.4875.

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Abstract Four CML patients were investigated for post transplant chimerism for total marrow (BM) cells and Mesenchymal Stem Cell (MSC) populations purified and propagated from BM cells. Patients were conditioned with BuFluATG and after allogeneic PBPCT were disease free with proven hematological, cytogenetical and genetical remission. For the study 15–30 ml fresh BM aspirates were phenotypically characterized for the presence of cell populations which may contain MSC. We found (median values): 11.7% CD45-CD34− 1.7% CD45-CD34-CD105+, 0.13% CD45-CD34-CD90+ and 0.03% CD45-CD34-CD73+ cells. These BM populations contained 0.75% CD34+ cells. Genetical work showed that BM cells were BCR-ABL negative and their STR informative allele patterns were consistent with those of donors. BM cells were processed as follows: incubation with Glycophorin A, CD3, CD14, CD19, CD66b, CD38 antibody cocktail which by cross-linking unwanted cells with red blood cells led to rosette formation (RosetteSep MSC Enrichment Cocktail, StemCell Technology), unrosetted cells (MSC enriched) were recovered from the interface after buoyant gradient centrifugation and contained (median values): 68.7% CD45-CD34-, 21.6% CD45-CD34-CD105+, 1.1% CD45-CD34-CD90+ and 0.4% CD45-CD34-CD73+. MSC enriched BM populations were cultured in Medium for Human MSC with Stimulatory Supplements (StemCell Technology). After 10–14 days CFU-F colonies were scored (median value was 77 CFU-F/106 cells) and cells were further cultured until >90% confluence of fibroblast-like cells were reached (usually 3 to 4 weeks after culture initiation). The cells were detached with 0.05% trypsin-EDTA and studied for STR allele patterns, the presence of BCR-ABL transcripts (at that time cells showed STR alleles of the recipient pattern for the first time - mixed chimerism) and the bulk of cells were further passaged. Usually after 3–4 passages (within 7–8 weeks) when fibroblast-like stromal cell populations reached the level of 3x106 cells, cultures were terminated and the cells were studied. These cells were in (median values) 27.0% CD45-CD34-, 23.8% CD45-CD34-CD105+, 26.5% CD45-CD34-CD90+ and 24.3% CD45-CD34-CD73+. The cells had a fibroblast-like morphology but only 26% had phenotype features of MSC on average. Therefore, the population consisted of MSC and more differentiated cells originated from CFU-F (MSC). RNA and DNA were isolated from the cells propagated for 7–8 weeks from the MSC enriched BM populations were BCR-ABL negative. However, their STR informative allele patterns were consistent with those of the recipients in variance to primary BM cell populations which was in all 4 cases of donor origin. Conclusions: with the use of the RosetteSep MSC enrichment purification system BM cells can be enriched in CFU-F which paralleled with an increase in the proportion of CD45-CD34-, CD45-CD34-CD90+, CD45-CD34-CD73+ and CD45-CD34-CD105+ cells, CFU-F BM enriched populations can be cultured with Medium for Human MSC with Stimulatory Supplements for successful propagation of fibroblast-like cells with kinetics documenting ex potential growth after 38 days (median) of culture, Cells originated from CFU-F were in contrast to the BM hematopoietic compartment of the recipients origin and were also BCR-ABL negative, MSC were not replaced by allogeneic PBPC-graft derived MSC.

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Suryawan, Nur, Lelani Reniarti, Hishamshah Ibrahim et Asohan Thevarajah. « Characteristics and Outcomes of Severe Aplastic Anemia Patients Who Were Treated with Allogenic Hematopoietic Stemcell Transplantation at Institute Pediatric Hospital Kuala Lumpur during 2003-2012 ». American Journal of Clinical Medicine Research 5, n^o 2 (26 août 2017) : 15–19. http://dx.doi.org/10.12691/ajcmr-5-2-1.

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Alcorn, M. J., T. L. Holyoake, L. Richmond, C. Pearson, E. Farrell, B. Kyle, D. J. Dunlop et al. « CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity. » Journal of Clinical Oncology 14, n^o 6 (juin 1996) : 1839–47. http://dx.doi.org/10.1200/jco.1996.14.6.1839.

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PURPOSE The objectives of this phase I study were to assess the feasibility of using cryopreserved peripheral-blood progenitor cells (PBPC) for large-scale CD34 selection and subsequent expansion, and the safety of their use for reinfusion following chemoradiotherapy. PATIENTS AND METHODS For 10 patients with nonmyeloid malignancy, an aliquot from a PBPC harvest was recovered from liquid nitrogen, and CD34 selected using the Isolex system (Baxter Healthcare, Newbury, United Kingdom) and expanded for 8 days ex vivo in a medium free of animal proteins but supplemented with autologous serum, stemcell factor (SCF), interleukin-1 beta (IL-1 beta), IL-3, IL-6, and erythropoietin. RESULTS The mean increase for cell number was 21-fold, for colony-forming units-granulocyte/macrophage (CFU-GM) 139-fold, and for burst-forming units-erythroid (BFU-E) 114-fold. The expanded cells were reinfused in tandem with unmanipulated material (> or = 25 x 10(4) CFU-GM/kg). The patients did not experience any adverse effects immediately on cell infusion or within 48 hours. The 10 index patients were compared with 10 historical controls for parameters of myelosuppressive morbidity. In this small study, there were no differences in either neutrophil or platelet recovery between the patients who received expanded cells and historical controls. CONCLUSION These data demonstrate that CD34 cells can successfully be selected from cryopreserved material, expanded ex vivo on a large scale, and safely reinfused following myeloablative conditioning regimens.

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Nakano, N., Y. Takatsuka, M. Tokunaga, S. Takeuchi, A. Kubota, M. Tokunaga et A. Utsunomiya. « Chronic GVHD After Allo-SCT Would Be Necessary For Better Outcome Of Allogenic Stemcell Transplantation (Allo-SCT) For Chemorefractory Non-Hodgkin's Lymphomas -A Single Institute Retrospective Study ». Biology of Blood and Marrow Transplantation 16, n^o 2 (février 2010) : S239. http://dx.doi.org/10.1016/j.bbmt.2009.12.256.

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Medeiros, Bruno C., Kathy Chun, Suzanne Kamel-Reid et Jeffrey Lipton. « Inv (11)(p15q21) in donor-derived Ph-negative cells in a patient with chronic myeloid leukemia in relapse successfully treated with imatinib mesylate post allogeneic stemcell transplantation ». American Journal of Hematology 82, n^o 8 (2007) : 758–60. http://dx.doi.org/10.1002/ajh.20882.

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Martin-Iglesias, Sara, Lara Milian, María Sancho-Tello, Rubén Salvador-Clavell, José Javier Martín de Llano, Carmen Carda et Manuel Mata. « BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models ». Stem Cells International 2022 (4 mars 2022) : 1–15. http://dx.doi.org/10.1155/2022/4910399.

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Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an in vitro osteogenic differentiation model based on mesenchymal stem cells (MSCs), to first analyse the influence of the culture media and the origin of the cells on the efficiency of this process and secondly to extrapolate it to a 3D environment to evaluate its possible application in bone regeneration therapies. Two osteogenic culture media were used (one commercial from Stemcell Technologies and a second supplemented with dexamethasone, ascorbic acid, glycerol-2-phosphate, and BMP-2), with human cells of a mesenchymal phenotype from two different origins: adipose tissue (hADSCs) and dental pulp (hDPSCs). The expression of osteogenic markers in 2D cultures was evaluated in several culture periods by means of the immunofluorescence technique and real-time gene expression analysis, taking as reference MG-63 cells of osteogenic origin. The same strategy was extrapolated to a 3D environment of polylactic acid (PLA), with a 3% alginate hydrogel. The expression of osteogenic markers was detected in both hADSCs and hDPSCs, cultured in either 2D or 3D environments. However, the osteogenic differentiation of MSCs was obtained based on the culture medium and the cell origin used, since higher osteogenic marker levels were found when hADSCs were cultured with medium supplemented with BMP-2. Furthermore, the 3D culture used was suitable for cell survival and osteogenic induction.

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Şerban, Georgiana Mihaela, Ion Bogdan Mănescu, Doina Ramona Manu et Minodora Dobreanu. « Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells ». Acta Medica Marisiensis 64, n^o 2 (1 juin 2018) : 83–90. http://dx.doi.org/10.2478/amma-2018-0011.

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Abstract Objective: Peripheral blood mononuclear cells (PBMC) are extremely important in the body’s immune response. Their isolation represents a major step in many immunological experiments. In this two phase study, we aimed to establish an optimum protocol for PBMC isolation by density-gradient centrifugation. Methods: During Phase-1, we compared two commercially available PBMC isolation protocols, Stemcell Technologies (ST) and Miltenyi Biotec (MB), in terms of PBMC recovery and purity. Twelve blood samples were assigned to each protocol. Each sample was divided in three subsamples of 1ml, 2ml and 3ml in order to assess the influence of blood sample volume on isolation performance. During Phase-2, a hybrid protocol was similarly tested, processing six blood samples. Additionally, we performed a flow cytometric analysis using an Annexin-V/Propidium-Iodide viability staining protocol. Results: Phase-1 results showed that, for all subsample volumes, ST had superior PBMC recovery (mean values: 56%, 80% and 87%, respectively) compared to MB (mean values: 39%, 54% and 43%, respectively). However, platelet removal was significantly higher for MB (mean value of 96.8%) than for ST (mean value of 75.2%). Regarding granulocyte/erythrocyte contamination, both protocols performed similarly, yielding high purity PBMC (mean values: 97.3% for ST and 95.8% for MB). During Phase-2, our hybrid protocol yielded comparable results to MB, with an average viability of 89.4% for lymphocytes and 16.9% for monocytes. Conclusions: ST yields higher cell recovery rates and MB excels at platelet removal, while the hybrid protocol is highly similar to MB. Both cell recovery and viability increase with blood sample volume.

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Burkhardt, Birgit, Alfred Reiter, Eva Landmann, Peter J. Lang, Lisa Lassay, Roswitha Dickerhoff, Max Lakomek, Guenter Henze et Arend von Stackelberg. « Poor Outcome for Children and Adolescents with Progressive Disease or Relapse of Lymphoblastic Lymphoma - a Report of the BFM Group ». Blood 112, n^o 11 (16 novembre 2008) : 3589. http://dx.doi.org/10.1182/blood.v112.11.3589.3589.

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Abstract Background/Objectives: Little is known about the outcome of pediatric patients with lymphoblastic lymphoma (LBL) who suffer form progressive disease or relapse. Patients and Methods: We analyzed the pattern of LBL-relapses after current NHL-BFM-frontline therapy between 4/90 and 3/03. Relapse therapy was according to ALL-Relapse-BFM protocols or ALL-BFM protocols for high-risk patients. Results: 28 of 251 registered T-LBL-patients (11%) and six of 73 pB-LBL-patients (8%) suffered from relapse. Of the 28 T-LBL-patients, one died from infection during relapse-chemotherapy, 18 failed to achieve stable remission and died from disease-progression, and nine reached allogeneic stemcell transplantation (SCT). Two of these nine SCT patients died from transplantation-associated toxicity, three died from disease-progression and four are still alive. The patients are in 2nd remission of their lymphoma for 48, 68, 125 and 131 months respectively after allogeneic SCT. One of the four patients developed colon adenocarcinoma 47 months after SCT. Of the six relapsed pB-LBL patients one died due to toxicity of relapse-chemotherapy, two died from disease-progression after chemotherapy and three received allogeneic SCT. Of these, two died from subsequent disease-progression while one patient is still alive 57 months after allogeneic SCT. Conclusions: Outcome of LBL-patients with relapse during or after current intensive 1st line therapy is poor. More than 50% of these patients failed to achieve remission to intensive 2nd line chemotherapy. Consolidation by allogeneic SCT may offer a cure for those who reach second remission.

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Rylova, Yu V., L. B. Buravkova et B. D. Zhivotovky. « The effects of cysplatin on human adipose tissue derived mesenchymal stromal cells under different oxygen levels ». Annals of the Russian academy of medical sciences 71, n^o 2 (6 février 2016) : 114–20. http://dx.doi.org/10.15690/vramn614.

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Objective: To evaluate the damaging effects of cisplatin on MMSCs from adipose tissue in a phase of active proliferation and the state of the monolayer, which was exposed at standard (20%) and reduced to 1% and 5% level of oxygen.Methods: The incubation MMSC with cisplatin was performed on cultures of 2 passage in a state in monolayer and cultures in the active growth phase. Profile surface markers of MMSC determined by flow cytometry. MMSCs viability after incubation with cisplatin was detected by the number of apoptotic and necrotic cells using ANNEXIN V-FITC - PI Kit (Immunotech, France). Standard culture conditions (~ 20% O2) created in a CO2 incubator (Sanyo, Japan), 5% O2 created using multigas incubator (Sanyo, Japan), 1% O2 - using an airtight chamber (Stemcell Technologies, USA).Results: Incubation of monolayer MMSC with cisplatin at a concentration of 10 ug/ml for 72 hours leads to death of half of the cells in culture under 20% O2, 5% O2 and 1% O2. Cisplatin increased the fracture of PI+-cell, and PI+/Ann+-cells under all culture conditions. The short-term exposure with cisplatin (24 and 48 hours) did not cause the damaging effect. Effects of cisplatin on the MMSC in the growth phase for 48 hours led to accumulation of Ann+-cells and PI+/Ann +-cells under all culture conditions. However least damaging effect of cisplatin was observed in culture under hypoxic conditions (1% O2).Conclusion: These data suggest that monolayer MMSCs are dying primarily through necrosis, whereas MMSC in the growth phase in response to cisplatin treatment are dying by apoptosis, regardless the oxygen tension.

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Monuki, Edwin S., Aileen J. Anderson, Mathew Blurton-Jones et Brian J. Cummings. « Response to StemCells Inc. » Stem Cell Reports 8, n^o 2 (février 2017) : 195–97. http://dx.doi.org/10.1016/j.stemcr.2017.02.002.

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Corona Lisboa, José Luis. « Biological importance of stemcells ». Journal of Stem Cell Research & ; Therapeutics 6, n^o 2 (21 avril 2020) : 70. http://dx.doi.org/10.15406/jsrt.2020.06.00143.

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This document is a short article of reflection on the biological importance of stem cells and the current state of therapeutic practices around these magnificent cells, physiologically speaking gifted. The article briefly describes the three main types of tissues for obtaining stem cells, together with the comments made by the author and other reference research on the topic.

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Abegunde, Samuel Ojo, et Michael J. Rauh. « Tet2-Deficient Bone Marrow Progenitors Have a Proliferative Advantage in the Presence of TNF-Alpha and IFN-Gamma : Implications for Clonal Dominance in Inflammaging and MDS ». Blood 126, n^o 23 (3 décembre 2015) : 2850. http://dx.doi.org/10.1182/blood.v126.23.2850.2850.

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Abstract Background: TET2 is a hematopoietic tumor suppressor gene that has been implicated in DNA demethylation and the epigenetic regulation of gene expression. Inactivating TET2 mutations are common in aging-associated clonal hematopoiesis of indeterminate potential (CHIP) and myelodysplastic syndromes (MDS). TET2 mutations may contribute to early clonal dominance and myeloid transformation, although the exact mechanisms remain to be elucidated. Emerging evidence suggests that the MDS bone marrow niche may be abnormal and this abnormal niche may act as fertile ground for expansion of neoplastic cells in vivo. Common to the environment of MDS and “inflammaging” are elevations in cytokines, such as TNFa and IFNg. We hypothesized that TET2 mutant clones may thrive in an inflammatory environment and further condition this environment to promote their own survival. Methods: Adult (10-14 weeks-old) Tet2 wild type and Tet2 mutant C57BL/6 mice strains (JAX) were chosen as a model system. The floxed Tet2 allele was deleted by targeting exon 3 with Vav1-cre mediated, hematopoietic-specific excision. We isolated lineage negative cells (Lin-), enriched for hematopoietic stem and progenitor cells (HSPC), from Tet2 wild type and -/- and bone marrow (BM) (EasySep; StemCell Technologies) and cultured these in the absence or presence of TNFα (0.1, 1, or 10 ng/ml) and IFN-γ (0.01, 0.1 or 1 ng/ml) in a methylcellulose colony formation assay (MethoCult; StemCell) or liquid culture media, and then examined their colony growth, cell count and phenotypic characterization over a period of 12 days. Where indicated, serial re-plating was performed. Results: We found an increased proportion of Lin- cells in Tet2 -/- BM compared to wild type, suggesting in vivo HSPC expansion. In triplicate experiments starting with equal numbers of wild type and Tet2 -/- Lin- cells (104 cells/MethoCult well), we found no significant difference in colony counts on days 3, 6, 9, or 12, when cultured in the absence or presence of increasing TNFα concentrations. As expected, TNFα dose-dependently reduced colony counts in both genotypes (up to 3 to 4-fold at 10 ng/ml). However, Tet2 -/- Lin- cells displayed a proliferative advantage over wild type in serial re-plating assays. In the presence of TNFα, this Tet2 -/- re-plating advantage was striking. As exemplified by day 12 colony counts at first re-plating (Fig. 1), while wild type colonies declined with increasing TNFα, Tet2 -/- colony counts increased with TNFα concentration (i.e. average 20-fold higher than wild type at 10 ng/ml TNFα; p<0.05). We next shifted our analysis to IFNg, and found significantly increased day 6, 9 and 12 Tet2 -/- methylcellulose colonies at first plating. Upon re-plating in IFNg, Tet2 -/- cells demonstrated significantly increased (1.5 to 2-fold; p<0.05) mean day 12 colony counts at 0.01, 0.1 and 1 ng/ml IFNg. To gain some insight into the nature of the cells emerging under IFNg stress, we performed flow cytometry upon re-plating. Preliminary experiments revealed increases (1.5 to 2-fold) in Mac1+Gr1+ and Sca1+Kit1+ populations in Tet2 -/-, as compared to wild type, in the presence of IFNg. We are currently comparing apoptosis in wild type and Tet2 -/- cells in the MethoCult system +/- TNFα and IFNg (and a more amenable liquid culture system), using Annexin V/Propidium Iodide-based flow cytometry. These results will be reported. Future directions include the characterization of differential: a) gene expression signatures in Tet2 wild type and -/- Lin- cells under TNFα and IFNg stress, and b) TNFα and IFNg signatures in TET2 -mutant and non-mutant human MDS. Conclusion: Tet2 -deficient murine bone marrow progenitors demonstrate a proliferative advantage, as compared to their wild type counterparts, under TNFα and IFNg stress. Given that these inflammatory cytokines have been associated with inflammaging and myelodysplasia, it is worth exploring whether TET2 -mutant human clones may emerge under inflammatory stress, leading to CHIP and/or MDS, and presenting a novel therapeutic target for clone eradication. Disclosures No relevant conflicts of interest to declare.

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Egeler, Oliver, Albertus W. Wognum, Caren Grande, Ning Yuan, Steven Woodside et Terry Thomas. « Automation of the Hematopoietic CFC Assay for Human Cord Blood, Bone Marrow and Mobilized Peripheral Blood Samples ». Blood 118, n^o 21 (18 novembre 2011) : 3001. http://dx.doi.org/10.1182/blood.v118.21.3001.3001.

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Abstract Abstract 3001 The hematopoietic colony-forming-cell (CFC) assay is a valuable tool to assess the potency of cell products (umbilical cord blood, apheresis products, bone marrow) for hematopoietic stem cell transplantation and for toxicity screening in therapeutic drug development. However, the manual colony enumeration that has been required is subjective and time consuming even for experienced users. This subjectivity limits the accuracy of the assay and contributes to a high degree of inter-laboratory variability. To reduce this variability, STEMCELL Technologies has developed an imaging and analysis system (STEMvision™) for automation of CFC assay colony enumeration. We have previously shown results validating the instrument for use in cord blood (CB) cell assays (Wognum et al. 37th Annual Meeting of the European Group for Blood and Marrow Transplantation, Hamburg, Germany 2011). Here we additionally present recent results comparing automated and manual colony counting for human mobilized peripheral blood (mPB), and bone marrow (BM). Samples of CB, mPB, and BM mononuclear cells were inoculated into semi-solid culture media (Methocult™ H4034, H4434, and H4435) and plated into special meniscus-free culture plates (SmartDish™). After 14 days in culture, automated colony counts were obtained from each sample using algorithms specifically optimized for each type of product (CB, mPB, and BM). The same cultures were then manually enumerated by 2–5 operators using the standard microscope method (microscope counts) and by enumerating colonies on the STEMvision™ images (image counts). For the each of the cell products (hCB, mPB, BM), the automated total colony counts were highly correlated to the average manual total colony counts. The table below compares the total manual image counts to the automated counts. Linear regression of the data shows that in addition to being highly correlated (r2 >0.90), the two counting methods give nearly identical results on average (the line of identity has a slope of 1). The efficacy of automated classification of colonies as erythroid or myeloid+mixed was evaluated by comparing the proportion of myeloid counts (myeloid / total) in each sample for image and automated counts. The % agreement was determined as as the differential between the myeloid proportions of the manual image and automated counts. The table below shows that on average, the agreement was greater than 90%. Variability of colony counting was also significantly reduced with the STEMvision™ instrument. We found that for multiple independent measurements of a given sample, the coefficient of variance (CV) of the normalized counts was 11% for the microscope counts (2–5 different operators), 7.9% for the image counts (2–5 different operators) and 4.4% for the automated counts (2–5 different instruments). The low CV for the automated counts was not operator dependent: for 6 samples analyzed by 6 operators using a single instrument, the CV for normalized total colony counts was 4.3%. Automated colony counting with the STEMvision™ instrument has thus been shown to be highly correlated to manual scoring methods for the most common hematopoietic stem cell transplantation products(CB, mPB, and BM). In addition, automation of the assay analysis significantly reduced the variability across multiple operators relative to manual counting methods. As a result, STEMvision™ has the ability to improve standardization of the CFC assay analysis through reduced intra- and inter-lab variability. We have reported previously on the internal and independent multi-center validation of this automated system for CB products. Rigorous validation of for BM and mPB cell products is currently in progress. In addition to providing standardization, this instrument reduces the time required for the assay readout and provides a means of permanent archiving of colony assay images. In the future, the image analysis output will provide quantitative information related to colony morphology that is not easily obtained by manual analysis (eg. colony size, density, symmetry). Such information would enable automated assessment of hematopoietic toxicity. Disclosures: Egeler: Stemcell Technologies: Employment. Wognum:Stemcell Technologies: Employment. Grande:Stemcell Technologies: Employment. Yuan:Stemcell Technologies: Employment. Woodside:Stemcell Technologies: Employment. Thomas:Stemcell Technologies: Employment, Patents & Royalties.

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Darche, Fabrice F., Nina D. Ullrich, Ziqiang Huang, Michael Koenen, Rasmus Rivinius, Norbert Frey et Patrick A. Schweizer. « Improved Generation of Human Induced Pluripotent Stem Cell-Derived Cardiac Pacemaker Cells Using Novel Differentiation Protocols ». International Journal of Molecular Sciences 23, n^o 13 (30 juin 2022) : 7318. http://dx.doi.org/10.3390/ijms23137318.

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Current protocols for the differentiation of human-induced pluripotent stem cells (hiPSC) into cardiomyocytes only generate a small amount of cardiac pacemaker cells. In previous work, we reported the generation of high amounts of cardiac pacemaker cells by co-culturing hiPSC with mouse visceral endoderm-like (END2) cells. However, potential medical applications of cardiac pacemaker cells generated according to this protocol, comprise an incalculable xenogeneic risk. We thus aimed to establish novel protocols maintaining the differentiation efficiency of the END2 cell-based protocol, yet eliminating the use of END2 cells. Three protocols were based on the activation and inhibition of the Wingless/Integrated (Wnt) signaling pathway, supplemented either with retinoic acid and the Wnt activator CHIR99021 (protocol B) or with the NODAL inhibitor SB431542 (protocol C) or with a combination of all three components (protocol D). An additional fourth protocol (protocol E) was used, which was originally developed by the manufacturer STEMCELL Technologies for the differentiation of hiPSC or hESC into atrial cardiomyocytes. All protocols (B, C, D, E) were compared to the END2 cell-based protocol A, serving as reference, in terms of their ability to differentiate hiPSC into cardiac pacemaker cells. Our analysis revealed that protocol E induced upregulation of 12 out of 15 cardiac pacemaker-specific genes. For comparison, reference protocol A upregulated 11, while protocols B, C and D upregulated 9, 10 and 8 cardiac pacemaker-specific genes, respectively. Cells differentiated according to protocol E displayed intense fluorescence signals of cardiac pacemaker-specific markers and showed excellent rate responsiveness to adrenergic and cholinergic stimulation. In conclusion, we characterized four novel and END2 cell-independent protocols for the differentiation of hiPSC into cardiac pacemaker cells, of which protocol E was the most efficient.

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