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Universal Platelets from induced Plueripotent Stem Cells : A Review Ojaswi B1, Anand B2 KIIT School of Biotechnology Bhubaneswar, Odisha 1, NIIH ICMR Mumbai Maharashtra2
INTRODUCTION Indications for platelet transfusion: 1. To treat thrombocytopenia 2. Platelet dysfunction
Production of HLA-KO iPLATS by knocking out β2 Microglobulin in imMKCL
imMKCL is re-re-programed to MK iPSC B2M knocked out using episomal vector from MK iPSC HLA-KO MK-iPSC is created
Limitation for platelet transfusion: 1. Less donors 2. Costly process 3. Transfusion transmission reactions
Doxycycline is added to makHLA-KO imMKCL, left to expand and mature
The degree of response depends upon the degree of disparity of HLA.
HLA-KO iPLAT released when Doxycycline is off
Use of iPSCs: Gene therapy Disease modelling Drug therapy
Establishment of a β2-Microglobulin-Knockout in imMKCL Use of a targeting vector plasmid, single-guide RNA (sg RNA) expression vector, episomal vector, re-reprogramming to establish MK-iPSC line #11, HLA-KO MK-iPSCs placed into cell co-culture condition Flow Cytometry For staining cells were incubated with 36 antibodies Plot retraction assay Mouse model At 10 weeks old, the mice were checked for the reconstitution of human hematopoietic cells Human stem cells were isolated and injected to mice and flow cytometry analysis was done Checked for IL-15 and Chimera protein.
RESULTS
Figure 4
The percentage of HLA-KO type among circulating iPLATs treated with anti-HLA-A2 antibody in Hu-NK-MSTRG mice
DISCUSSION Whether platelets catch B2M in circulation to express functional HLA-I is unknown. The generation of anti-HPA alloantibodies is the second leading cause of allo-PTR and also the main cause of fetal/ neonatal alloimmune thrombocytopenia and post-transfusion purpura.
CONCLUSIONs & FUTURE GOALS
in vitro cytotoxic assays of NK Cells co-cultered with iPLATS or imMKCLs
NK Cells collected from Healthy donors
Stem Cell Reports.2020;14(1):49-59. Figure 4
HLA-KO iPLATS began circulating efficiently in a Humanized Mouse model with High NK Cell Reconstitution
iPLATS or K562 cells co-cultured with Human IL-2 MATERIALS AND METHODS
Stem Cell Reports.2020;14(1):49-59.
REFERENCES 1.
Analysis by Flow Cytometry
We came across this article “iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity” which had these OBSERVATIONS keywords: “induced pluripotent stem cells”, “Natural Killer Cells”, “Human Leukocyte Antigen Cell Culture Class I”,“ Platelet Transfusion”. This was published DOX-OFF production of iPLATs condition: the medium contained 10% human plasma in the Stem Cell Reports having an impact factor of MK-iPSCs established from imMKCLs by 5.499 . This article was looked in to with other re-reprogramming articles in PubMEd Research.
To be successful in making universal platelets To evaluate the outcome of the study in human trials. Studies are done till stage 3 and the results are satisfactory. Ensure long-term survival of the platelet transplant To make platelets easily availability
Suzuki D, Flahou C, Yoshikawa N, et al. iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity. Stem Cell Reports. 2020;14(1):49-59.
ACKNOWLEDGEMENTs Stem Cell Reports.2020;14(1):49-59. Figure 4
number of total circulating hCD41a+CD42b+ iPLATs and percentage of HLA-KO type among circulating iPLATs in Hu-NK-MSTRG mice at pre-transfusion and 30 min, 3 h, and 6 h after transfusion
CSIR SRTP -2020 Biotechnika Daisuke Suzuki, Charlotte Flahou, Norihide Yoshikawa -Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto,Japan