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Abhijeet Sethi
Abhijeet Sethi

This six-month progress report summarizes work done on a project aiming to profile microRNA expression during hepatic differentiation from human umbilical cord Wharton's jelly-derived mesenchymal stem cells. The objectives accomplished so far include obtaining necessary approvals and collecting 12 human umbilical cords from which Wharton's jelly was isolated without enzymes using an explant method. The isolated Wharton's jelly-derived mesenchymal stem cells were maintained in culture. Future work will involve differentiating the cells into hepatocyte-like cells and profiling microRNA expression at different stages of differentiation.

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MicroRNA (miRNA) Expression Profiling during Hepatic Differentiation from Stem Cells Six Monthly Progress Report (March 2013) SVKMs NMIMS (Deemed to be University) SUBMITTED BY AkshataKishorRaut GUIDED BY Dr.AparnaKhanna School of Science SVKMs NarseeMonjee Institute of Management Studies, (Deemed-to-be University), V. L. Mehta Road, Vile-Parle (West), Mumbai 400056.
PROJECT TITLE: MicroRNA (miRNA) Expression Profiling during Hepatic Differentiationfrom Stem Cells RATIONALE OF THE STUDY: MicroRNAs (miRNA) are key regulators involved in various important biological processes including the regulation of cell cycle, differentiation and development yet, the role of miRNAs at stage of cellular proliferation while differentiating at early developmental phases, particularly in human liver, is unknown. There has been one study wherein comparative gene and miRNA expression analysis in adult liver tissues and embryonic liver samples was performed, hence Systematic screening of miRNAs expressed in developing hepatic cells is essential to understand miRNArole in hepatogenesis. Aim: The current project aims to find out miRNA expression profiling during hepatic differentiation from human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs). Also, the mRNA and miRNA expression patterns will be studied using taqman assays. This research would provide insights regarding role of miRNA during hepatic differentiation. Efforts will be made to determine whether stage specific miRNAs are seen, which can be used as a biomarker for a particular stage of differentiation. Hence umbilical cord cells can be work as excellent model system to mimic the in vivo differentiation process. Research reports shows that percentage of HLCs derived from hWJ-MSCs is very low hence this project also aims to develop stage wise procedure to increase percentage of HLCs The ultimate aim of the project, beyond the scope of the present proposal would be to demonstrate the role of a particular detected miRNA, which might be involved in the regulation of biological pathway (during differentiation) by functional analysis, through translational repression or degradation of specific mRNA targets.
OBJECTIVES OF THE STUDY: Isolation, maintenance and characterization of human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs) Standardization of protocols for differentiation of hepatocyte-like cells (HLCs) 1. Molecular characterization 2. Cellular characterization 3. Functional characterization MicroRNA expression profiling OBJECTIVES PROPOSED AND DISCUSSED IN FIRST TAC MEETING Isolation and maintenance of human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs). OBJECTIVES ACCOMPLISHED IN LAST SIX MONTHS: A. Collection of Human Umbilical Cords: Institutional Ethics Committee (IEC) and Institutional Committee on Stem Cell Research & Therapy (IC-SCRT) approvals were obtained for collection of human umbilical cords (hUCs). Further, necessary approvals from Dr. BalabhaiNanavati Hospital Ethics Committee were taken. With the consent of the parents fresh hUCs (n=12) were aseptically collected from infants delivered full term by normal labor at Dr.BalabhaiNanavati Hospital, Mumbai.After collection, umbilical cords were stored aseptically at 40 C in Dulbecco`s Phosphate Buffered Saline (DPBS) containing 1% antibiotics until processing. B. Isolation and maintenance of human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs): For this study, hWJ-MSCs were isolated without enzymatic treatment i.e. by culturing Whartons jelly (gelatinous connective tissue) and by using Ex-plant method. i. Isolation Protocol for Method 1 (Scraping of Whartons jelly): Cords were collected in DPBS containing 2% antibiotics. Following disinfection in 70% ethanol for 30Secs again cord was transferred into falcon containing 20 -25 ml of DPBS (shake it properly to get rid of traces of ethanol as well as blood). Then cord was transferred into petri plate containing DPBS. Cord was cut in pieces of 5cm, and each section was squeeze slightly with scalpel to remove unwanted blood clots which were
stuck inside the vessels. For isolation of Whartons jelly cord was cut into small pieces (about 1.5cm length), which was sectioned longitudinally to expose whartons jelly and jelly was scraped off with scalpel. Jelly was collected in 10-15ml of complete medium (10% FBS+ DMEM) and centrifuged at 1600rpm for 10 minutes to get the cell pellet. Supernatant was discarded and cell pellets re-suspended in DMEM culture medium supplemented with 10% FBS. Total yield was plated into one T-75 flask.

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Dc report rough

  • 1. MicroRNA (miRNA) Expression Profiling during Hepatic Differentiation from Stem Cells Six Monthly Progress Report (March 2013) SVKMs NMIMS (Deemed to be University) SUBMITTED BY AkshataKishorRaut GUIDED BY Dr.AparnaKhanna School of Science SVKMs NarseeMonjee Institute of Management Studies, (Deemed-to-be University), V. L. Mehta Road, Vile-Parle (West), Mumbai 400056.
  • 2. PROJECT TITLE: MicroRNA (miRNA) Expression Profiling during Hepatic Differentiationfrom Stem Cells RATIONALE OF THE STUDY: MicroRNAs (miRNA) are key regulators involved in various important biological processes including the regulation of cell cycle, differentiation and development yet, the role of miRNAs at stage of cellular proliferation while differentiating at early developmental phases, particularly in human liver, is unknown. There has been one study wherein comparative gene and miRNA expression analysis in adult liver tissues and embryonic liver samples was performed, hence Systematic screening of miRNAs expressed in developing hepatic cells is essential to understand miRNArole in hepatogenesis. Aim: The current project aims to find out miRNA expression profiling during hepatic differentiation from human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs). Also, the mRNA and miRNA expression patterns will be studied using taqman assays. This research would provide insights regarding role of miRNA during hepatic differentiation. Efforts will be made to determine whether stage specific miRNAs are seen, which can be used as a biomarker for a particular stage of differentiation. Hence umbilical cord cells can be work as excellent model system to mimic the in vivo differentiation process. Research reports shows that percentage of HLCs derived from hWJ-MSCs is very low hence this project also aims to develop stage wise procedure to increase percentage of HLCs The ultimate aim of the project, beyond the scope of the present proposal would be to demonstrate the role of a particular detected miRNA, which might be involved in the regulation of biological pathway (during differentiation) by functional analysis, through translational repression or degradation of specific mRNA targets.
  • 3. OBJECTIVES OF THE STUDY: Isolation, maintenance and characterization of human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs) Standardization of protocols for differentiation of hepatocyte-like cells (HLCs) 1. Molecular characterization 2. Cellular characterization 3. Functional characterization MicroRNA expression profiling OBJECTIVES PROPOSED AND DISCUSSED IN FIRST TAC MEETING Isolation and maintenance of human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs). OBJECTIVES ACCOMPLISHED IN LAST SIX MONTHS: A. Collection of Human Umbilical Cords: Institutional Ethics Committee (IEC) and Institutional Committee on Stem Cell Research & Therapy (IC-SCRT) approvals were obtained for collection of human umbilical cords (hUCs). Further, necessary approvals from Dr. BalabhaiNanavati Hospital Ethics Committee were taken. With the consent of the parents fresh hUCs (n=12) were aseptically collected from infants delivered full term by normal labor at Dr.BalabhaiNanavati Hospital, Mumbai.After collection, umbilical cords were stored aseptically at 40 C in Dulbecco`s Phosphate Buffered Saline (DPBS) containing 1% antibiotics until processing. B. Isolation and maintenance of human umbilical cord Whartons jelly derived mesenchymal stem cells (hWJ-MSCs): For this study, hWJ-MSCs were isolated without enzymatic treatment i.e. by culturing Whartons jelly (gelatinous connective tissue) and by using Ex-plant method. i. Isolation Protocol for Method 1 (Scraping of Whartons jelly): Cords were collected in DPBS containing 2% antibiotics. Following disinfection in 70% ethanol for 30Secs again cord was transferred into falcon containing 20 -25 ml of DPBS (shake it properly to get rid of traces of ethanol as well as blood). Then cord was transferred into petri plate containing DPBS. Cord was cut in pieces of 5cm, and each section was squeeze slightly with scalpel to remove unwanted blood clots which were
  • 4. stuck inside the vessels. For isolation of Whartons jelly cord was cut into small pieces (about 1.5cm length), which was sectioned longitudinally to expose whartons jelly and jelly was scraped off with scalpel. Jelly was collected in 10-15ml of complete medium (10% FBS+ DMEM) and centrifuged at 1600rpm for 10 minutes to get the cell pellet. Supernatant was discarded and cell pellets re-suspended in DMEM culture medium supplemented with 10% FBS. Total yield was plated into one T-75 flask.