Blood products are used to treat a multitude of diseases, so the blood transfusion system needs to be enhanced. CRISPR/Cas9 has made it viable to make HLA class I-deleted blood products to avoid rejection

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Blood Transfusion ◽  
Blood Donation ◽  
Es Cells ◽  
Scale Up ◽  
Embryonic Stem ◽  
Hla Class I ◽  
Class I ◽  
Blood Products ◽  
Expiry Date ◽  
Ips Cell

In adults, normal hematopoiesis occurs in the bone marrow, producing leukocytes, red blood cells, and platelets. Recently, megakaryocytes have been found in mouse lungs and spleen, where they release platelets by blood flow force. Blood products are used to treat a multitude of diseases and conditions that generate cytopenia. The blood transfusion system must be enhanced due to a drop in blood donors due to low birth rate and changing attitudes among young people, pathogen contamination, and rising demand due to chronic blood diseases that are prevalent among the elderly. Pluripotent stem cells, such as embryonic stem (ES) cells, may proliferate in vitro indefinitely and are a prospective source for blood transfusions to replace blood donations.Platelet preparations can be maintained at room temperature to sustain platelet function, but only have a statutory expiry date of five days. Platelets are anucleate cells, thus irradiation before blood donation can lessen the risk of iPS cell infection. Effective treatment requires HLA-compatible platelet transfusions, although supply limits often leave patients underserved. CRISPR/Cas9 has made it viable to make HLA class I-deleted blood products to avoid rejection and lower the odds of platelet-expressed human leukocyte antigen Class I cancer-causing iPS cells (HLA-I). This article discusses the production of megakaryocyte cell lines, bioreactors, and scale-up cultures, as well as identifying viable drugs in manufacturing. HLA-null, iPSC-derived platelet products' universal potential will also be explored.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

An analysis of the adaptive immune response towards an embryonic stem cell grafts

2007 ◽  
Vol 30 (4) ◽  
pp. 98
Author(s):  
Douglas Wu ◽  
Kathryn Wood
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Adaptive Immune Response ◽  
Class I ◽  
Es Cell ◽  
Mhc Class I Molecule

Background: Although clinical transplantation has had enormous impact on the treatment of premature organ failure, shortage of donor organs continues to be a crucial limiting factor. Embryonic stem cells represent an attractive potential source of replacement tissue because of their inherent pluripotentiality and ability to self-renew. However, before any ES cell-based cellular replacement strategies can be considered, many issues must be addressed. Among these is an evaluation of the potential immune response elicited by any ES cell graft. Because ES cells express very low levels of MHC class I and no MHC class II, their immunogenicity has been questioned. Here we utilize a BM3 TCR transgenic model to analyze the adaptive immune response against an ES cell graft in vivo. Methods: BM3 CD8 TCR-tg T cells (H2K background) specific for the MHC class I molecule H2Kb were labelled with CFSE and adoptively transferred into CBA rag recipients. The following day, ES cells derived from a CBA, B6, or CBK background were implanted beneath the kidney capsule of adoptively transferred mice. Response of the CD8 T cells was measured via CSFE division profiling and graft infiltration. Results: CFSE division profile of naïve BM3 CD8 T cells was unaltered by the presence of either a syngeneic or an allogeneic ES cell graft. These naïve cells were also unable to recognize and infiltrate either a syngeneic or allogeneic ES cell graft on days 5 and 10 post-implantation, despite strong expression of the MHC class I molecule H2Kb by engrafted allogeneic ES cells. On the other hand, H2Kb+ islets begun to be infiltrated by day 5, and were obliterated by a vigorous allogeneic response by day 10. When H2Kb+ islets were implanted into the same kidney as allogeneic ES cells (opposite poles), islet grafts were rapidly infiltrated by CD4 and CD8 T cells and destroyed, but ES cell grafts exhibited markedly reduced cellular infiltrate. In contrast to naïve BM3 CD8 T cells, however, activated cells recognized and mounted an aggressive cytotoxic response against an allogeneic ES cell graft which could be detected by day 6 and resulted in complete graft destruction by day 10. Conclusions: Under certain circumstances, an ES cell graft may have reduced immunogenicity as compared with other conventional tissue or solid organ allografts. This may be due to their lack of passenger APC, which may in turn cripple their ability to elicit a robust allogeneic response via the direct pathway of allorecognition. However, because of their strong upregulation of allogeneic MHC class I molecules after transplantation, they are still likely to elicit a significant rejection response when transplanted into recipients replete with both CD4 and CD8 T cells.

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3846-3846
Author(s):  
Ji-Yoon Noh ◽  
Shilpa Gandre-Babbe ◽  
Yuhuan Wang ◽  
Vincent Hayes ◽  
Yu Yao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitors ◽  
Transfusion Therapy ◽  
Ips Cell

Abstract Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Immunobiology of naive and genetically modified HLA-class-I-knockdown human embryonic stem cells

2011 ◽  
Vol 124 (23) ◽  
pp. 4127-4128 ◽  
Author(s):  
T. Deuse ◽  
M. Seifert ◽  
P. S. Tsao ◽  
X. Hua ◽  
J. Velden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Genetically Modified ◽  
Embryonic Stem ◽  
Hla Class I ◽  
Class I

scholarly journals COVID-19 pandemic: Challenges and approaches in blood transfusions

2020 ◽  
Keyword(s):  
Blood Transfusion ◽  
Blood Donation ◽  
Virus Transmission ◽  
Blood Transfusions ◽  
Blood Products ◽  
Screening Programs ◽  
Self Sufficiency ◽  
Comprehensive Information ◽  
Bacterial Transmission ◽  
Donated Blood

One of the goals of the Iranian Blood Transfusion Organization is to provide adequate healthy blood and reduce the risk of various viral and bacterial transmission infections. With the removal of alternative blood donation, all blood units and blood products are provided through voluntary blood donation in Iran. The Blood Transfusion Organization screens the donated blood according to standard guidelines to ensure blood recipients and physicians of providing healthy and pathogen-free components. With the emergence of novel pathogens, such as the pandemic of the COVID-19 virus, despite Iranchr('39')s self-sufficiency in blood supply and blood products, the number of blood donors has decreased significantly since there is a lack of comprehensive information on pathophysiology and virus transmission ways. Moreover, the existence of some shortages in screening programs can cause problems. Therefore, this study was performed to review the studies conducted investigating this emerging virus regarding blood transfusions and the supply of blood components worldwide.

Large Scale Generation of Functional Megakaryocytes From Human Embryonic Stem Cells (hESCs) Under Stromal-Free Conditions.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2540-2540
Author(s):  
Feng Li ◽  
Shi-Jiang Lu ◽  
Qiang Feng ◽  
Robert Lanza
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Es Cells ◽  
Scale Up ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Immunofluorescent Staining ◽  
Promising Alternative ◽  
Human Es Cells

Abstract Abstract 2540 Poster Board II-517 Platelets collected from donors have very limited shelf life and are increasingly needed for transfusions. In contrast to donor dependent cord blood or bone marrow CD34+ stem cells, hESCs are a promising alternative source for continuous in vitro production of platelets under controlled conditions. Current procedures for in vitro generation of megakaryocytes/platelets from hESCs are not efficient and require undefined animal stromal cells. We have developed a novel system to generate megakaryocytes (MKs) from human ES cells under serum and stromal-free conditions. In the current system, hESCs are directed towards megakaryocytes through distinct steps including embryoid body formation and hemangioblast development (Lu et al, Nature Methods, 4:501–509, 2007). A transient bi-potential cell population expressing both CD41a and CD235a markers has been identified at the end of hemangioblast culture. These cells are capable of generating both MKs and erythroid cells as demonstrated by FACS sorting and CFU assays. TPO, SCF and IL-11 are used to further direct MK differentiation of hemangioblasts derived from human ES cells in suspension culture. Currently, up to 2.5×107 MKs (CD41a+) can be generated from 1×106 hESCs, which is approximately 10 times more efficient than recently reported methods (Takayama et al Blood, 111(11):5298–5306, 2008). Without further purification, >90% of live cells from the suspension cultures are CD41a+ and the majority of these cells are also CD42b+ (>70%). These in vitro derived MK cells have morphological characteristics of mature, polyploid MKs as shown by Giemsa staining and immunofluorescent staining of vWF in cytoplasmic granules. Importantly, proplatelet forming cells are constantly observed at the late stage of MK culture indicating that MKs generated in this system are able to undergo terminal differentiation under feeder-free conditions. Platelet-like particles are also detected in culture media by FACS. When plated on OP9 cells, these MKs generate functional platelets that are responsive to thrombin stimulation. In summary, we have established a novel system for the generation of platelet-producing MKs from human ES cells that is suitable for scale up and future preclinical and clinical studies. Disclosures: Li: Stem Cell & Regenerative Medicine International: Employment. Lu:Stem Cell & Regenerative Medicine International: Employment. Feng:Stem Cell & Regenerative Medicine International: Employment. Lanza:Stem Cell & Regenerative Medicine International/Advanced Cell Technology, Inc: Employment.

Implementing Haemovigilance in India as a National Perspective

2020 ◽  
Vol 7 (1) ◽  
pp. 30-36
Author(s):  
Vignesh Mano ◽  
Raman S. Kumar
Keyword(s):  
Blood Transfusion ◽  
Adverse Reactions ◽  
Blood Donation ◽  
National Level ◽  
Developed Countries ◽  
Quality And Safety ◽  
Blood Products ◽  
National Perspective ◽  
The Developed Countries ◽  
Indian Perspective

Haemovigilance is an organized and effective process of monitoring, identifying, reporting, investigating and analyzing adverse events and reactions in case of blood transfusion and during the manufacturing process of blood products. This system ensures the quality and safety aspects of blood transfusion, that bring out corrective and preventing actions and advancement in the transfusion system. Nowadays, most of the developed countries have implemented Haemovigilance in order to monitor adverse reactions and events associated with blood donation and transfusion. This review article is about steps that are required to be taken for the implementation of Haemovigilance on a National level as an Indian perspective.

scholarly journals Research Progress and Application Prospect of IPS Cells

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Ceng Yiwu
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Ips Cells ◽  
New Drugs ◽  
Theoretical Research ◽  
Research Progress ◽  
Ips Cell

Differentiated somatic cells can be reprogrammed into induced pluripotent stem cells (iPS cells) by introducingspecific transcription factors. This technique avoids immune rejection and ethical problems in stem cell research.A great revolution in the fi eld of science. As with embryonic stem cells (ES cells), iPS cells are able to self-renewand maintain undiff erentiated state. In vitro, iPS cells can be induced to diff erentiate into a variety of mature cells,therefore, iPS cells in theoretical research and clinical applications are extremely valuable. IPS cell diff erentiationand transplantation in the treatment of blood diseases have a great use, iPS cells can treat nervous system diseases,to provide in vitro disease model, to study the mechanism of disease formation, screening new drugs and thedevelopment of new to provide a new treatment The The use of iPS cells as a nuclear donor cell, with the appropriatereceptor cells after fusion can be directly obtained transgenic animals. Not only can improve the genetic nature ofanimals, but also can break the boundaries of species and get the new animal traits that cannot achieve by usingtraditional mating methods. The research of iPS cells has been widely concerned, and it is the research hotspot in cellbiology and molecular biology. In this paper, the defi nition of iPS cells, the acquisition of iPS cells, the history ofdevelopment, the signifi cance of research, the progress of research, the application of iPS cells, and the problems ofiPS cells were reviewed.

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