In Vitro Culture of Rat Primary Hepatocytes Employing Monolayer and Spheroid Culture Environments

Imran Ullah,Yeongji Kim,Malgum Lim,Keon Bong Oh,Seongsoo Hwang,Yurianna Shin,Youngim Kim,Gi-Sun Im,Tai-Young Hur,Sun A Ock 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

Primary hepatocytes (PH) are considered as “gold standard” for drug screening because of its ability to express the entire set of drug metabolizing enzymes and transporters. Hepatocytes culturing and maintaining their hepatocyte fate in vitro is one of the major issue from last decade. The main problem in hepatocytes in vitro culturing is that, these cells rapidly loss their hepatic morphology and liver specific function in culture condition. In the present study, we isolated rat PH and cultured in monolayer (2D) as well as spheroid (3D) culture system. The 2D cultured PH hepatocyte showed an elongated hepatocyte morphology while, 3D cultured PH showed spheroid morphology with gradual decrease in diameter up to 7 days. After 7 days of in vitro culture, these cells were analyzed for the expression of hepatic markers (Alb, Tf, Afp) and apoptotic markers (Bax, Bcl2). Furthermore, PH in both culture systems was induced with two inducers i.e. 3-methylcholanthrene (3-MC, Cyp1a specific inducer) and dexamethasone (Cyp3a specific inducer) for 48 and 72 hours, respectively. The mRNA level of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PH, respectively. After 7 days in vitro culture, PH showed dramatic down regulation of hepatic markers in both culture systems. Furthermore, expression of apoptotic markers was higher in 2D cultured PH as compared to 3D. Cyp1a and Cyp3a mRNA level showed higher RNA content in 2D culture PH after 48 of induction. Therefore, we concluded that there was no significant difference found in two culture system and further studies are needed to find out the essential components for PH in vitro culture rather than culture system.

Small Molecules-Driven Pancreatic ß-Cells Differentiation of Bone Marrow Mesenchymal Stem Cells

Imran Ullah,Keon Bong Oh,Yurianna Shin,Youngim Kim,Sun A Ock 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Diabetes mellitus, a hyperglycemic condition, in which the patients either fail to secrete insulin because of ß-cells destruction (type I) or shows insulin resistance (type II), affecting more than 300 million people around the globe. Generation of effective ß-cells for the treatment of diabetes is a key area in modern translational medicines. Here we tried to generate porcine induced pancreatic ß-cells (piPan ß-cells) from Gal- TKO+MCP porcine bone marrow mesenchymal stem cell (pBM-MSCs) by overexpressing set of transcription factors included EGFP along with step-wise induction of small molecules. pBM-MSCs were cultured in media with 5-azacytidine for 24 hours and then proceeded with transfection using episomal transfection system. Transfected cells were replaced with basal media containing growth factors cocktail, Activin A and 2 uM of A83-01 followed by retinoic acid treatment. Finally, cells were replaced by maintenance media until maturation. After 3 days of transfection, EGFP expression was observed showing successful transfection which disappeared completely after 21 days. pBM-MSCs attained definitive endoderm morphology after Activin A treatment and formed exact pancreatic clusters after 4 weeks and 8 weeks in maintenance media, respectively. After 4 and 8 weeks, piPan ß-cells were analyzed for the expression of pancreatic markers by immunofluorescence (Insulin, PAX6, Somatostatin) and RT-qPCR (GCG, INS, NKX6.1, PDX1 and NEUROD1), which showed significantly higher expression as compared with pBM-MSCs. Morphological changes and expression of pancreatic markers revealed successfully differentiation of PiPan ß-cells; however, more in depth studies for its functional characteristic and in vivo maintenance are still in progress.

Protective function of nicotinamide against ketamine-induced apoptotic neurodegeneration in the infant rat brain.

Ullah, Najeeb,Ullah, Ikram,Lee, Hae Young,Naseer, Muhammad Imran,Seok, Park Moon,Ahmed, Jawad,Kim, Myeong Ok Birkhäuser Boston 2012 Journal of molecular neuroscience Vol.47 No.1

<P>During development, anesthetics activate neuroapoptosis and produce damage in the central nervous system that leads to several types of neurological disorders. A single dose of ketamine (40?mg/kg) during synaptogenesis in a 7-day-old rat brain activated the apoptotic cascade and caused extensive neuronal cell death in the forebrain. In this study, we investigated the protective effect of nicotinamide against ketamine-induced apoptotic neurodegeneration. After 4?h, neuronal cell death induced by ketamine was associated with the induction of Bax, release of cytochrome c into the cytosol, and activation of caspase-3. One single dose of 1?mg/g nicotinamide was administered to a developing rat and was found to inhibit ketamine-induced neuroapoptosis by downregulating Bax, inhibiting cytochrome c release from mitochondria into cytosol, and inhibiting the expression of activated caspase-3. TUNEL and immunohistochemical analyses showed that ketamine-induced cell death occurred through apoptosis and that it was inhibited by nicotinamide. Fluoro-Jade-B staining demonstrated an increased number of dead cells in the cortex and thalamus after ketamine treatment; treatment with nicotinamide reduced the number of dead cells in these brain regions. Our findings suggest that nicotinamide attenuated ketamine-induced neuronal cell loss in the developing rat brain and is a promising therapeutic and neuroprotective agent for the treatment of neurodevelopmental disorders.</P>

The Impact of Capital Account Openness on Income Inequality: Empirical Evidence from Asia

Imran ULLAH,Fayaz Hussain TUNIO,Zia ULLLAH,Agha Amad NABI 한국유통과학회 2022 The Journal of Asian Finance, Economics and Busine Vol.9 No.2

The relationship between income inequality and capital account openness is empirically investigated in this study, where macroeconomic variables have opposing effects. Panel data used in the study from the KAOPEN Index and World Bank consists of 28 Asian countries and has been examined; it contains annual observations from 1970 to 2018. The data is examined using a random-effect model based on GMM estimates. Income inequality and capital account openness are positively and significantly related, according to our findings. Overall, the findings imply that increasing income gaps reduced capital investment in nations with large discrepancies. The growing economic discrepancy is being caused by the rich’s increasing income share at the expense of the poor. In Asia, inward capital account openness exacerbates income inequality, while outward capital account openness exacerbates it. As a result, income inequality slows economic growth, leading to inflation, unemployment, and increased government spending in several Asian countries. Our control factors, GDP, and other secondary school enrolments, all had a statistically significant negative relationship with income inequality. Income disparity has a positive and statistically significant association with government spending, inflation, population, trade openness, and unemployment. Income disparity has a negative association with capital account openness, gross domestic product, and secondary school enrollment.

Human mesenchymal stem cells - current trends and future prospective

Ullah, Imran,Subbarao, Raghavendra ,Baregundi,Rho, Gyu ,Jin Portland Press Ltd. 2015 Bioscience reports Vol.35 No.2

<▼1><P>Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term <I>in vitro</I> culturing, <I>in vitro</I> differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.</P></▼1><▼2><P>In this review, we highlighted recent research findings in the area of human mesenchymal stem cells, its application in the treatment of chronic diseases and its use in human clinical trials.</P></▼2>

Transdifferentiation of α-1,3-galactosyltransferase knockout pig bone marrow derived mesenchymal stem cells into pancreatic β-like cells by microenvironment modulation

Ullah Imran,Lee Ran,Oh Keon Bong,황성수,Kim Youngim,Hur Tai-Young,Ock Sun A 아세아·태평양축산학회 2020 Animal Bioscience Vol.33 No.11

Objective: To evaluate the pancreatic differentiation potential of α-1,3-galactosyltransferase knockout (GalTKO) pig-derived bone marrow-derived mesenchymal stem cells (BM-MSCs) using epigenetic modifiers with different pancreatic induction media. Methods: The BM-MSCs have been differentiated into pancreatic β-like cells by inducing the overexpression of key transcription regulatory factors or by exposure to specific soluble inducers/small molecules. In this study, we evaluated the pancreatic differentiation of GalTKO pig-derived BM-MSCs using epigenetic modifiers, 5-azacytidine (5-Aza) and valproic acid (VPA), and two types of pancreatic induction media – advanced Dulbecco's modified Eagle's medium (ADMEM)-based and N2B27-based media. GalTKO BM-MSCs were treated with pancreatic induction media and the expression of pancreas-islets-specific markers was evaluated by real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. Morphological changes and changes in the 5'-C-phosphate-G-3' (CpG) island methylation patterns were also evaluated. Results: The expression of the pluripotent marker (POU class 5 homeobox 1 [OCT4]) was upregulated upon exposure to 5-Aza and/or VPA. GalTKO BM-MSCs showed increased expression of neurogenic differentiation 1 in the ADMEM-based (5-Aza) media, while the expression of NK6 homeobox 1 was elevated in cells induced with the N2B27-based (5-Aza) media. Moreover, the morphological transition and formation of islets-like cellular clusters were also prominent in the cells induced with the N2B27-based media with 5-Aza. The higher insulin expression revealed the augmented trans-differentiation ability of GalTKO BM-MSCs into pancreatic β-like cells in the N2B27-based media than in the ADMEM-based media. Conclusion: 5-Aza treated GalTKO BM-MSCs showed an enhanced demethylation pattern in the second CpG island of the OCT4 promoter region compared to that in the GalTKO BM-MSCs. The exposure of GalTKO pig-derived BM-MSCs to the N2B27-based microenvironment can significantly enhance their trans-differentiation ability into pancreatic β-like cells.

In vitro 3-D culture demonstrates incompetence in improving maintenance ability of primary hepatocytes

Imran Ullah,옥선아,김영지,임맑음,오건봉,황성수,Yurianna Shin,김영임,임기선,허태영 한국통합생물학회 2017 Animal cells and systems Vol.21 No.5

Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-Dcultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72 h, respectively. The mRNA levels of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PHs. After 7 days of in vitro culture, PHs exhibited dramatic downregulation of hepatic marker expression in both cultures. Furthermore, apoptotic marker expression was higher in the 2-D-cultured PHs than 3-D-cultured PHs. The mRNA levels of Cyp1a and Cyp3a indicated higher RNA content in the 2-D-cultured PHs after 48 h of induction. Therefore, we concluded that there was no significant difference between the culture systems, and further studies are required to identify the essential components for in vitro PH culture rather than culture systems.

Transplantation of Human Dental Pulp-Derived Stem Cells or Differentiated Neuronal Cells from Human Dental Pulp-Derived Stem Cells Identically Enhances Regeneration of the Injured Peripheral Nerve

Ullah, Imran,Park, Ju-Mi,Kang, Young-Hoon,Byun, June-Ho,Kim, Dae-Geon,Kim, Joo-Heon,Kang, Dong-Ho,Rho, Gyu-Jin,Park, Bong-Wook Mary Ann Liebert 2017 STEM CELLS AND DEVELOPMENT Vol.26 No.17

Induction of the differentiation of porcine bone marrow mesenchymal stem cells into premature hepatocyte-like cells in an indirect coculture system with primary hepatocytes

Ullah Imran,서강민,위하연,김영민,이승훈,옥선아 한국통합생물학회 2020 Animal cells and systems Vol.24 No.5

Liver transplantation is currently the only option for patients with end-stage liver disease. Thus, other alternate therapeutic strategies are needed. Bone marrow mesenchymal stem cells (BMMSCs) are nonhematopoietic cells present in the bone marrow stroma that serve as precursors cells for various other cells. In this study, we evaluated the differentiation of porcine BM-MSCs into hepatocyte-like cells using three types of culture systems: hepatic induction medium (HIM), HIM/primary hepatocyte culture supernatant (HCS; 1:1 ratio), and a hepatocyte coculture system (HCCS; primary hepatocytes in the upper chamber, and BM-MSCs in the lower chamber). Primary hepatocytes were isolated from anesthetized healthy 1-month-old pigs by enzymatic digestion. Hepatic-specific marker expression (albumin [ALB], transferrin [TF], α-fetoprotein [AFP]), glycogen storage, low-density lipoprotein, and indocyanine green uptake were evaluated. Upregulation of hepatic-specific markers (ALB, TF, and AFP) was observed by real-time polymerase chain reaction in the HCCS group. Periodic acid-Schiff staining revealed enhanced glycogen storage in hepatocyte-like cells from the HCCS group compared with that from the HIM/HCS group. Furthermore, hepatocyte like-cells in the HCCS group showed improved LDL and ICG uptake than those in the other groups. Overall, our current study revealed that indirect coculture of primary hepatocytes and BM-MSCs enhanced the differentiation efficacy of BM-MSCs into hepatocyte-like cells by unknown useful soluble factors, including paracrine factors.

Decreased GABABR expression and increased neuronal cell death in developing rat brain after PTZ-induced seizure.

Naseer, Muhammad Imran,Ullah, Ikram,Al-Qahtani, Mohammed H,Karim, Sajjad,Ullah, Najeeb,Ansari, Shakeel Ahmed,Kim, Myeong Ok,Bibi, Fehmida Springer-Verlag Italia 2013 Neurological sciences Vol.34 No.4

<P>The objective of this study was to evaluate the PTZ-induced seizures effects on GABAB receptor (R) expression and to observe its neurodegenerative effect in hippocampal part of developing rat brain. In the present study, high dose of pentylenetetrazol (PTZ 40 mg/kg) was injected in developing rats of age 5 weeks having average weight of 60-65 g for 4 days. Further, baclofen (B 3 mg/kg i.p) agonist and phaclofen (P 30 μg/rat) antagonist of GABABR were injected along with PTZ. Western blot analysis was used to elucidate expression of GABABR protein upon PTZ, baclofen and phaclofen exposure in the developing rat brain. Furthermore, PTZ-induced apoptotic neurodegeneration was also observed through the release of caspase-3 antibody and propidium iodide (PI) staining using confocal microscopy. Seizure was confirmed using electroencephalography (EEG) data obtained from the Laxtha EEG-monitoring device in the EEG recording room and EEG was monitored 5-15 min after PTZ injection. The results of the present study showed that PTZ-induced seizure significantly decreased GABABR expression and induced neuronal apoptosis in cortical and hippocampal part of brain. While, baclofen reverse the effect of PTZ by increasing the expression of GABABR as compared to the PTZ- , PTZ plus B- and PTZ plus P-treated groups. Our findings indicated that PTZ-induced seizure showed not only decrease in GABABR expression but also cause neuronal apoptosis in the developing rat brain.</P>