Additional file 3: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S1. showing sequencing results of parental and inserted iPSCs. a Parental iPSCs have the known F9 gene mutation c.676C > T, p.Arg226Trp. b Inserted iPSCs (colony 5) have a heterozygous mutation of c.676C > T, p.Arg226Trp. (DOCX 393 kb

Additional file 1: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Table S1. presenting antibodies used for immunofluorescence staining and flow cytometry analysis. (DOCX 14 kb

Additional file 7: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S4. showing characterization of hepatocytic functions. Differentiated cells had functions of glycogen storage (a) and ICG uptake (b), and also expressed LDL-receptor (c) and had ability for LDL uptake (d). All scale bars represent 100 Îźm. (DOCX 1747 kb

Additional file 2: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Table S2. presenting primers used for characterization of hepatocytic functions. (DOCX 14 kb

Additional file 5: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S2 showing characterization of iPSC colony 5. a Karyotype of iPSC colony 5 was normal. b qRT-PCR analysis showed expression of OCT4, SOX2, and NANOG of iPSC colony 5. PBMNCs of patient used as negative control, H1 embryonic stem cells used as positive control. c Immunofluorescence staining showed expression of TRA-1-60, SSEA4, OCT4, and NANOG. d Sections of teratomas stained with H&E (endoderm: pancreas; mesoderm: muscle; ectoderm: nerve fibers). All scale bars represent 100 Îźm. (DOCX 1261 kb

Additional file 6: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Figure S3. showing off-target effects detection in successful inserted iPSCs. Using Cas-OFFinder, 1799 potential off-target sites that differed from the sgRNA sequence by up to five nucleotides in the genome were found. We found 97,968 indels, 3084 SVs, 51,628 SNPs, and 2225 CNVs unique to the inserted iPSCs compared to that in the parental iPSCs. Since indels and SVs comprise virtually all of the mutations introduced by CRISPR-Cas9, we focused solely on indels and SVs. Through comparison of potential off-target sites, and indels and SVs unique to the inserted iPSCs, we found no overlapping mutation between them. (DOCX 302 kb

Additional file 4: of Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system

Table S3 presenting plasmids used for transfection of HEK293T cells and iPSCs. (DOCX 14 kb

Recombinant human thrombopoietin (rhTPO) of different dosing regimens for refractory/relapsed primary immune thrombocytopenia: a multicenter, randomized controlled trial and pharmacokinetics study

Recombinant human TPO (rhTPO) is effective for refractory/relapsed primary immune thrombocytopenia (ITP), but optimal dosing regimen remains elusive. In this multicenter, randomized, controlled trial, a total of 282 adult ITP patients (mean age 47.3 years; 82 men) with a platelet count ≤30 × 109/L or >30 × 109/L with active bleeding randomly received a once daily (QD) subcutaneous injection of 7500 U (n = 64) or 15000 U rhTPO for 14 injections, or 15000 U or 30000 U rhTPO once every other day (QOD) for 7 injections. The primary outcomes included change from baseline in platelet count and total response rate (TRR) on day 14. On day 14, the median increase of platelet count from baseline was the highest in the 15000-U QD group (167.5 × 109/L, interquartile range [IQR] 23.0–295.0 × 109/L), followed by the 30000-U QOD group (57.5 × 109/L, IQR 9.0–190.0 × 109/L) (ANCOVA P P = .266 with baseline count as a covariate). The TRR on day 14 was also the highest in the 15000-U QD group (63.2%), followed by the 30000-U QOD group (59.7%). The rate of grade 3 and above adverse events did not differ among the four groups. There were no new safety concerns. All 4 regimens are safe and well-tolerated. The 30000-U QOD regimen is practically indistinguishable in efficacy to the 15000-U QD regimen. What is the context?Relative thrombopoietin deficiency is implicated in primary immune thrombocytopenia (ITP), which is characterized by increased platelet destruction and impaired megakaryopoiesis.Patients who are innately unresponsive to or have relapsed after glucocorticoid treatment have limited treatment options.Recombinant human thrombopoietin (rhTPO) improves treatment response of primary ITP patients when added to high-dose dexamethasone. Relative thrombopoietin deficiency is implicated in primary immune thrombocytopenia (ITP), which is characterized by increased platelet destruction and impaired megakaryopoiesis. Patients who are innately unresponsive to or have relapsed after glucocorticoid treatment have limited treatment options. Recombinant human thrombopoietin (rhTPO) improves treatment response of primary ITP patients when added to high-dose dexamethasone. What is new?This trial sought to identify an optimal dosing regimen of rhTPO for patients who had failed or relapsed after glucocorticoid therapy.Of the 4 regimens, once daily 15000 U rhTPO for 14 injections yielded the greatest median increase in platelet count (167.5 × 109/L) from baseline and attained the highest total response rate on day 14 (63.2%).30000 U rhTPO once every other day for 7 injections was effective in rapidly increasing platelet counts in the first 7 days.All 4 regimens were safe and well-tolerated. This trial sought to identify an optimal dosing regimen of rhTPO for patients who had failed or relapsed after glucocorticoid therapy. Of the 4 regimens, once daily 15000 U rhTPO for 14 injections yielded the greatest median increase in platelet count (167.5 × 109/L) from baseline and attained the highest total response rate on day 14 (63.2%). 30000 U rhTPO once every other day for 7 injections was effective in rapidly increasing platelet counts in the first 7 days. All 4 regimens were safe and well-tolerated. What is the impact?The 30000 U rhTPO once every other day regimen may offer an effective and safe regimen with less frequent injections, but future trials with longer follow-up are needed. The 30000 U rhTPO once every other day regimen may offer an effective and safe regimen with less frequent injections, but future trials with longer follow-up are needed.</p