ABSTRACT
Platelets play a role in hemostasis in vivo, and platelet transfusion is the main means to treat bleeding diseases caused by thrombocytopenia or platelet dysfunction. However, platelets are in short supply due to the increasing demand for platelet products in clinical, the limited number of blood donors and the disadvantages of platelet products such as short shelf life and bacteria contamination. Currently, induced pluripotent stem cells are considered an ideal source for producing platelets in vitro. They have the potential for self-renewal and differentiation into any cell type, and can be obtained and manipulated easily. Given the recent advances in megakaryocytic series, bioreactors, feeder-free cell production and large-scale propagation research, platelet preparations derived from induced pluripotent stem cells have gradually shown great potential for clinical applications. Considering the minimal risk of alloimmunization and tumorigenesis with these blood products, they are promising to become the standard source of future blood transfusions. This paper reviews the research progress of the methodological techniques of in vitro generation of platelets from induced pluripotent stem cells.
ABSTRACT
【Objective】 To optimize the existing spin-EB method and promote human induced pluripotent stem cells (hiPSCs) differentiate into megakaryocytes (MKs). 【Methods】 In this study, the initial inoculation amount of hiPSCs was increased from 3 500 cells/well to 8 000 cells/well, and the size of EB was increased. By observing the generation time of EB- hematopoietic cells during differentiation, and detecting the proliferation of CD34+ hematopoietic progenitor cells and CD41+ MKs in different stages, it was studied whether the optimized scheme could promote the differentiation of hiPSCs into hematopoietic progenitor cells(HPCs) and MKs. 【Results】 By increasing the initial inoculation amount of hiPSCs and the size of EB, the differentiation of hiPSCs into HPCs and MKs and the cell production efficiency can be promoted. 【Conclusion】 Our research describes an optimized and repeatable differentiation method, which can produce hematopoietic progenitor cells and mature MKs from hiPSCs in a relatively short time with higher yield. It is of great clinical significance and broad scientific research prospect to continuously optimize the culture scheme of hiPSCs differentiation to produce MKs and platelets in vitro, and to promote large-scale platelet generation in vitro in transfusion medicine.
ABSTRACT
Platelet transfusion is the main clinical treatment of thrombocytopenia. However, due to the difficulty of platelet collection, high cost of use and limited number of blood donors, the development of platelet treatment is greatly limited. Therefore, the research on thrombogenesis in vitro has attracted more attention at home and abroad. Platelet production in vitro has the advantages of donor-independence, platelet antigen free and low risk of alloimmunity. At present, the efficiency of producing functional platelets in vitro is low, and there is still a big gap to achieve the ultimate goal of producing a large number of functional platelets in vitro. This paper reviews the research progress of megakaryocyte / platelet production in vitro, focuses on the in vitro production potential of megakaryocyte / platelet, and summarizes the current platelet culture systems in vitro based on human pluripotent stem cells, embryonic stem cells and adipose stem cells. The contradictions and difficulties of platelet production in vitro were also discussed to provide theoretical support for further research.
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【Objective】 To investigate the effect of cold agglutination on blood group typing. 【Methods】 37℃ water bath, absorption elution test and 2-mercaptoethanol method were used to eliminate the influence of cold agglutination. Forward and reverse blood group typing, cross matching, DAT and IAT experiments were then performed on red blood cells and serum after treatment. 【Results】 Before treatment, obvious discrepancy in forward /reverse typing and nontypable cross matching in 16 blood samples were noticed due to cold agglutination. After corresponding treatments, all samples were consistent or negative in forward/reverse typing, cross matching and antibody screening. No adverse reactions to cross matching blood transfusion occurred in patients, and the increase of hemoglobin was in line with the effective standard of transfusion. 【Conclusion】 37℃ water bath, absorption elution test and 2-mercaptoethanol method can be used to eliminate the interference caused by cold agglutination to obtain correct typing results. The strong reactivity caused by cold agglutination in AIHA patients were different from other cases, which deserved our attention.
ABSTRACT
Objective To explore the clinical efficacy of ultrasound - guided injection of platelet-rich plasma (PRP) for the treatment of recalcitrant lateral epicondylitis. Methods From September 2014 to June 2016, a total of 15 patients with recalcitrant lateral epicondylitis received ultrasound - guided injection of autologous PRP therapy, including via left arm injection (n=2) and via right arm injection (n=13). By using twice centrifugal method, the patient' s own venous whole blood was centrifuged to obtain PRP. All patients underwent PRP injection once a week, a total of 3 treatments were performed for each patient. Results After the first injection of PRP, the patients were followed up for 12 months. One month after the treatment, visual analogue scale (VAS) score was obviously improved, at 3 months after the treatment the improvement of VAS score reached its peak and it remained at this level until 12 months after the treatment. The elbow joint function, which was evaluated with modified MAYO elbow score, was also significantly improved in one month after the treatment, and the clinical effect was sustained to 12 months after the treatment. Conclusion Ultrasound - guided precise injection of PRP can effectively improve the pain and the elbow joint function caused by recalcitrant lateral epicondylitis. (J Intervent Radiol, 2018, 27:238-241)
ABSTRACT
<p><b>OBJECTIVE</b>To improve the MigR1-CD19-CAR (chimeric antigen receptor) that contains a single chain variable region (scFv) which targeted to CD19 through a retroviral vector transduction efficiency of T-lymphocytes.</p><p><b>METHODS</b>Insert the CD19-CAR fragment into the retroviral vector (MigR1) through recombinant DNA technology, after transfecting plat-A packaging cell lines, viral supernatant was collected to transduce K562 cell line and activated human T-lymphocytes. We used flow cytometry to determine the transduction efficiency and RT-PCR to confirm the transcription of CD19-CAR gene. The ability of the transduced T cells to produce IFN-γ and TNF-α in a CD19-specific manner was measured in an enzyme-linked immunosorbent (ELISA) assay.</p><p><b>RESULTS</b>(1)Using MigR1-CD19-CAR retroviral vector to produce the high titer retrovirus. (2)MigR1-CD19-CAR transduction efficiency of K562 cell line was significantly higher than human T-lymphocytes (P<0.01). (3)120 min centrifugation could significantly improve transduction efficiency of T-lymphocytes to (54.5±14.6)%. (4)Transduction efficiency could be improved by deciding transduce time according to T-lymphocytes proliferation fold in vitro individually, and the highest transduction efficiency in the study was 69.3%. The CD19-CAR gene sequence was transcripted specificly with high efficiency. (5) IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased to (13 230±1 543) pg/ml and (4 217±211) pg/ml when coculture with CD19-K562 cells.</p><p><b>CONCLUSION</b>We have successfully constructed a second generation CAR which targeted to CD19 through a retroviral vector called MigR1 (MigR1-CD19-CAR). Deciding transduce time according to T-lymphocytes proliferation fold in vitro individually and 120 min centrifugation could improve the CAR transduction efficiency of T-lymphocytes. RT-PCR confirmed that the CD19-CAR gene was specificly transcripted with high efficiency. IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased when activated by target cells.</p>