Induced pluripotent stem cells: Generation methods and a new perspective in COVID-19 research

Induced pluripotent stem cells (iPSCs) exhibit an unlimited ability to self-renew and produce various differentiated cell types, thereby creating high hopes for both scientists and patients as a great tool for basic research as well as for regenerative medicine purposes. The availability and safety of iPSCs for therapeutic purposes require safe and highly efficient methods for production of these cells. Different methods have been used to produce iPSCs, each of which has advantages and disadvantages. Studying these methods would be very helpful in developing an easy, safe, and efficient method for the generation of iPSCs. Since iPSCs can be generated from somatic cells, they can be considered as valuable cellular resources available for important research needs and various therapeutic purposes. Coronavirus disease 2019 (COVID-19) is a disease that has endangered numerous human lives worldwide and currently has no definitive cure. Therefore, researchers have been rigorously studying and examining all aspects of COVID-19 and potential treatment modalities and various drugs in order to enable the treatment, control, and prevention of COVID-19. iPSCs have become one of the most attractive and promising tools in this field by providing the ability to study COVID-19 and the effectiveness of drugs on this disease outside the human body. In this study, we discuss the different methods of generation of iPSCs as well as their respective advantages and disadvantages. We also present recent applications of iPSCs in the study and treatment of COVID-19

Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1

Objective: Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. Materials and Methods: We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. Results: After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. Conclusion: MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation

Is there any relationship between red blood cell distribution width and prognosis of brain death?

Background: Accumulating evidence has demonstrated that RDW (red blood cell distribution width) may independently predict clinically important outcomes in many populations. However, the role of RDW has not been elucidated in brain death. We conducted this study with the aim of evaluating the predictive value of RDW in brain death. Methods: A retrospective study of seventy-seven of brain death cases during 36 months were evaluated at university hospitals, affiliated in Tehran, Iran. Demographical data include age, sex, BMI and cause of brain death, also laboratory results (red blood cell distribution, mean corpuscular volume, hemoglobin) collected by checklists from patient records. Having the three RDW measurements (days of hospital admission, day of brain death, and day of cardiac arrest) required. Results: Time interval from hospital admission until brain death was 5.27±4.07. The mean age of brain death cases was 32.65±16.53. The mean RDW values on days of hospital admission, the day of brain death, and the day of cardiac arrest were 14.53±1.98, 15.12±1.93 and 15.18±2.07, respectively. Results of the repeated-measures ANOVA test reveal that RDW level was constantly higher in the traumatic patient group compared to the non-traumatic ones (P=0.008). Conclusion: The frequency of brain death was high in patients with high RDW values. RDW might be a prognostic biomarker for brain death. More prospective studies with large sample size and long follow-up period should be carried out to determine the prognostic significance of RDW and brain death in future

Expression of definitive endoderm markers.

<p>qRT-PCR analysis of definitive endoderm markers Sox-17 and HNF-3 beta/FoxA2 during differentiation on days 7, 14 and day 21 to islet-like clusters as compared with undifferentiated hMSCs (D0), the highest expression of both genes were detected on day 7. The expression levels were normalized to human GAPDH. The data are presented as mean±SEM. P <0.05.</p

Detection of pancreatic endocrine proteins levels by western blot in IPCs.

<p>Western blot analysis detected expressions of insulin (5KD), glucagon (46KD), PDX1 (31KD) and Ngn3 (23KD) in differentiated IPCs (1) MSCs^miR-375 and (2) MSCs^{miR-375+anti-miR-9} (3) MSCs^control.</p

Detection of Mtpn and OC-2 proteins levels by western blot in IPCs.

<p>Granuphilin: (A) MSCs^{miR-375+anti-miR-9} (B) MSCs^miR-375 (C) Control hMSCs, OC-2: (A) Control hMSCs (B) MSCs^anti-miR-9 (C) MSCs^{miR-375+anti-miR-9}. Mtpn: (A) Control hMSCs (B) MSCs^miR-375 (C) MSCs^{miR-375+anti-miR-9}. Protein level of Mtpn was markedly reduced after miR-375 up-regulation. However, a protein level of OC-2 was increased following anti-miR-9 infections after 21 days.</p

Expression of pancreatic endocrine genes in derived IPCs.

<p>The expression levels of pancreatic transcription factors such as <i>PDX1</i> and <i>Ngn3</i>, endocrine markers, <i>insulin</i>, <i>glucagon</i>, <i>somatostatin</i> and <i>PPY</i> and β cells specific genes like <i>Nkx2</i>.<i>2</i> and <i>GLUT2</i> were analyzed at each stage of differentiation into IPCs. Gene transcripts of IPCs were compared with undifferentiated hMSCs^null (negative control) and <i>PANC-1</i> cell line (positive control). Relative levels of gene expression were normalized to human <i>GAPDH</i>. Transcript value is shown in each graph as mean±SEM. * P <0.05.</p

hMSCs purification and infection.

<p>(A) The results of miR-375 and anti-miR-9 transduction examined by fluorescent microscopy (X100). (B) Transduction efficiency of MSCs was about 50% in comparison to control MSCs as determined by flow cytometry. The experiment was repeated at least three times.</p

Characterization of undifferentiated hMSCs.

<p>(A) Flow cytometric analysis of hMSCs surface markers at passage 3, the cells was strongly positive for CD105 and CD90 while negative for CD34 and CD31. (B) The cells were exposed to lineage specific media and adipogenic and osteogenic differentiation was confirmed by (A) Oil-red (X100) and (B) Alizarin-red staining respectively (X100).</p