Generation of an EYS-associated retinitis pigmentosa patient-derived human pluripotent stem cell line (MUi038-A).

Mutations in the eyes shut homolog (EYS) gene are one of the common causes of autosomal recessive retinitis pigmentosa (RP). The lack of suitable animal models hampers progress understanding of the disease mechanism and drug development. This study reported the reprogramming of CD34+ hematopoietic stem/progenitor cells from a patient with compound heterozygous EYS mutations (c.6416 G > A and c.7228 + 1 G > A) into the induced pluripotent stem cell line, MUi038-A, using non-integrating vectors. The MUi038-A demonstrates pluripotency, tri-lineage differentiation potential, and a normal karyotype, offering a valuable model for studying the mechanism of EYS-related RP and new therapeutic development.

A generation of human-induced pluripotent stem cell line (MUi032-A) from a Choroideremia disease patient carrying a hemizygous mutation on the CHM gene.

Choroideremia (CHM) is a monogenic, X-linked inherited retinal disease caused by mutations in the CHM gene. CHM patients develop progressive loss of vision due to degeneration of cell layers in the retina. In this report, the human-induced pluripotent stem cell, MUi032-A, was generated from CD34+ hematopoietic stem/progenitor cells of a male CHM patient by co-electroporation of non-integration episomal vectors containing OCT4/shp53, Sox-2/KLF4, and L-MYC/LIN-28. The MUi032-A showed normal karyotype and a hemizygous c.715C > T mutation. They expressed pluripotency markers and differentiated into cells derived from three germ layers. This cell line may be useful for disease mechanisms and gene therapy studies.

Intravitreal autologous mesenchymal stem cell transplantation: a non-randomized phase I clinical trial in patients with retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is a progressive inherited retinal disease with great interest for finding effective treatment modalities. Stem cell-based therapy is one of the promising candidates. We aimed to investigate the safety, feasibility, and short-term efficacy of intravitreal injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) in participants with advanced stage RP. METHODS: This non-randomized phase I clinical trial enrolled 14 participants, categorized into three groups based on a single dose intravitreal BM-MSC injection of 1 × 106, 5 × 106, or 1 × 107 cells. We evaluated signs of inflammation and other adverse events (AEs). We also assessed the best corrected visual acuity (BCVA), visual field (VF), central subfield thickness (CST), and subjective experiences. RESULTS: During the 12-month period, we noticed several mild and transient AEs. Interestingly, we found statistically significant improvements in the BCVA compared to baseline, although they returned to the baseline at 12 months. The VF and CST were stable, indicating no remarkable disease progression. We followed 12 participants beyond the study period, ranging from 1.5 to 7 years, and observed one severe but manageable AE at year 3. CONCLUSION: Intravitreal injection of BM-MSCs appears to be safe and potentially effective. All adverse events during the 12-month period required observation without any intervention. For the long-term follow-up, only one participant needed surgical treatment for a serious adverse event and the vision was restored. An enrollment of larger number of participants with less advanced RP and long-term follow-up is required to evaluate the safety and efficacy of this intervention. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01531348 . Registered on February 10, 2012.

Multicenter validation of fully automated capillary electrophoresis method for diagnosis of thalassemias and hemoglobinopathies in Thailand.

Thalassemias and hemoglobinopathies are highly prevalent in Thailand and other Southeast Asian countries. Accurate and precise separation of hemoglobin types, together with reliable quantitation, are essential for differential diagnosis of these diseases. Presented in this study is a multicenter validation of a fully automated capillary electrophoresis (CE) method for hemoglobin separation and quantitation involving four reference laboratories in Thailand. Analytical performance characteristics, including precision and accuracy were compared with existing validated HPLC and LPLC methods using 412 blood samples from unrelated subjects. Coefficient of variance of Hb A2 quantitation was 1.80-2.86, 1.26-5.13 and 1.08-6.66% for within run, between run and interlaboratory comparison, respectively. Results of Hb A2 and Hb F quantitated by the CE method correlates well with those of the two comparative methods (r = 0.98-0.99). The CE method correctly determined the genotypes (thalassemias and hemoglobin variants) of all blood samples tested. The major advantage of the CE system is its ability to separate and quantitate Hb A2, Hb E, Hb F, Hb H and Hb Bart's, which are important parameters required for diagnosis of thalassemias and hemoglobinopathies.

Validation of osmotic fragility test and dichlorophenol indophenol precipitation test for screening of thalassemia and Hb E.

The strategy for screening of thalassemia and Hb E by a combination of osmotic fragility (OF) test and dichlorophenol indophenol precipitation (DCIP) test was validated with 436 unrelated Thai subjects. Hemoglobin (Hb) typing, Hb A2 quantitation, PCR and DNA sequence analysis were used as confirmatory methods for diagnosis of thalassemia and hemoglobinopathy. The sensitivity and specificity of this strategy was 100% and 79.7%, respectively. The results assessed by two medical scientists were exactly the same with 93.3% accuracy in comparison with the confirmatory methods. A combination of OF and DCIP has been shown to be a reliable, rapid, simple and sensitive strategy for screening thalassemia and Hb E in the Thai population.

Prevention of thalassemia: experiences from Samui Island.

Thalassemia is one of the most important genetic disorders in Thailand. A model for prevention of thalassemia by combining 4 strategies including education, carrier screening, counseling and prenatal diagnosis was developed by a thalassemia task force. A thalassemia work group was formed in order to develop a mechanism to integrate the model for real use on Samui Island where thalassemia is common and a specific disease oriented program is required. 200 health professionals working on Samui Island participated in the thalassemia educational courses. The specific training courses were also provided for obstreticians, medical technologists and counselors. A team of well-trained health professionals was established to manage public education. Information booklets, posters, brochures and mass media including spot radio and newspapers were used as educational materials. For carrier screening, blood samples of pregnant women from all health care organizations on Samui Island were collected and screened for thalassemia carriers by using the osmotic fragility (OF) test/the dichlorophenol indophenol precipitation (DCIP) tests at Samui Hospital. Samples with positive results were sent to the Regional Medical Sciences Center in Surat Thani for thalassemia diagnosis. When a carrier was identified, her spouse was offered testing. For at-risk couples, details of the disorder were included in counseling to help them reach a decision that was right to them, in the context of their unique medical, moral and social situations. Amniotic fluid samples were collected for prenatal diagnosis. All patient information was registered by using computer software. After 1 year of integration by using facilities of the Ministry of Public Health service system, a wide range of problems were identified. They underlined the need for effective health service structure co-operation, adequate education of responsible health professionals, explicit policies and a clear line of responsibility at local, regional and national levels for service development and quality management. It is hoped that all information conducted in the present study will be useful to health authorities to develop an explicit policy and promote the health service structure co-operation in the country that will finally lead to successfully reducing the frequency of severe thalassemia in the future.

Hb G Makassar (beta 6:Glu-Ala) in a Thai family.

Hemoglobin G Makassar was identified in a family of Thai origin, which was found to share identical properties with hemoglobin S in routine hemoglobin separation both by cellulose acetate electrophoresis and cation-exchange HPLC. It is, therefore, subjects with hemoglobin G Makassar and hemoglobin S who may sometimes be mistakenly identified for each other. In this study, identification of hemoglobin G Makassar was achieved by DNA sequence analysis, which revealed a single nucleotide substitution GAG-->GCG at codon 6 of the beta-globin gene. The hemoglobin variant was hemoglobin G Makassar [beta 6:Glu-->Ala]. This is the first report of hemoglobin G Makassar in Thailand.

The validation of an automated liquid chromatography system for the diagnosis of thalassemias and hemoglobinopathies.

Accurate and precise hemoglobin separation and the quantitation of Hb A2 and Hb F are essential for the diagnosis of the thalassemias and hemoglobinopathies. Presented in this study is the validation of the the Hb A2 assay of the HbGold analyzer, a fully automated liquid chromatography system for hemoglobin separation and quantitation. Variability of Hb A2 quantitation was quite low; the CV's of within-run, between-run and interlaboratory studies were 1.8-3.1%, 3.4-6.0% and 6.8-8.8% respectively. The results of the %Hb A2 quantitated by HbGold analyzer correlated well with those given by the Bio-Rad Variant Hb testing system (r=0.98). The application of the HbGold analyzer for the diagnosis of the thalassemia phenotypes frequently observed in Thailand is considered. In conclusion, the Hb A2 assay of the HbGold analyzer could be used for the quantitation of Hb A2 and Hb F and the presumptive identification of abnormal hemoglobins.