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HISTOPATHOLOGICAL ANALYSIS OF PATIENTS WITH
ABRUPTIO PLACENTA IN A TERTIARY HEALTH CARE CENTRE

Dissertation submitted in
Partial fulfillment of the regulations required for the award of
M.D. Degree in
PATHOLOGY - BRANCH III

THE TAMILNADU
DR.M.G.R.MEDICAL UNIVERSITY
CHENNAI MAY 2019
DECLARATION

I hereby declare that the dissertation entitled “HISTOPATHOLOGICAL

ANALYSIS OF PATIENTS WITH ABRUPTIO PLACENTA IN A TERTIARY HEALTH CARE CENTRE” is a bonafide research work done

by me in the Department of Pathology, Coimbatore Medical College during the period from JANUARY 2017 TO JUNE 2018 under the guidance and

supervision of Dr.G.S.THIRIVENI BALAJJI, M.D, Associate Professor,

Department of Pathology, Coimbatore Medical College.
This dissertation is submitted to The Tamilnadu Dr.MGR Medical
University, Chennai towards the partial fulfillment of the requirement for the award of M.D., Degree (Branch III) in Pathology. I have not submitted this dissertation on any previous occasion to any University for the award of any Degree.

Place : Coimbatore Date :

Dr. A. PETER SAMIDOSS
CERTIFICATE

This is to certify that dissertation entitled “HISTOPATHOLOGICAL

ANALYSIS OF PATIENTS WITH ABRUPTIO PLACENTA IN A

TERTIARY HEALTH CARE CENTRE” is a bonafide work done by

Dr. A. PETER SAMIDOSS, a postgraduate student in the Department of Pathology, Coimbatore Medical College, Coimbatore under guidance and

supervision of Dr.G.S.THIRIVENI BALAJJI, M.D, Associate Professor,

Department of Pathology, Coimbatore Medical College, Coimbatore in partial fulfillment of the regulations of the Tamil Nadu Dr. M. G. R. Medical University, Chennai towards the award of M.D. Degree (Branch III) in Pathology.

  • Guide
  • Head of the Department

Dr.G.S.THIRIVENI BALAJJI, M.D,

Associate Professor,

Prof. Dr. C. LALITHA, M.D.,

Professor and Head,
Department of Pathology, Coimbatore Medical College, Coimbatore -14.
Department of Pathology, Coimbatore Medical College, Coimbatore -14.

Prof. Dr. B.ASOKAN, M.S, M.Ch.,

Dean, Coimbatore Medical College, Coimbatore -14.

ACKNOWLEDGEMENT

To begin with, I thank the almighty God for bestowing his blessing on me in this dissertation to be a successful one.

I wish to thank our beloved Dean Prof. Dr.B.ASOKAN,M.S.,M.Ch,

Coimbatore Medical College and Hospital, Coimbatore for permitting me to conduct this study.

I thank Prof. Dr. C.LALITHA, M.D., Professor and Head of the

Department, Department of Pathology, Coimbatore Medical College, Coimbatore for her guidance and support.
I wish to express my gratitude and sincere thanks to my guide

Dr.G.S.THIRIVENI BALAJJI, M.D, Associate Professor, Department of

Pathology, Coimbatore Medical College, Coimbatore. This dissertation bears her valuable suggestions and highly professional advice.

My heartful thanks to Dr.S.YOGALAKSHMI, M.D, Assistant

Professor, Department of Pathology, Coimbatore Medical College, Coimbatore for her timely advice and suggestion through the course of my work.
I thank all my Associate Professors and Assistant Professors of
Department of Pathology, Coimbatore Medical College, Coimbatore for their opinion and encouragement.

I thank Department of Obstetrics &Gynecology, Coimbatore Medical

College, Coimbatore, for providing clinical cases, valuable support and guidance which made this dissertation possible. I thank all my patients for their co-operation and support.
My deepest and most heart whelming thanks goes to my parents, my

father Mr.P.S.AYYAPILLAI and my mother Mrs.A.PADMAVATHY who

have showered me with lots and lots of love.

My special thanks to my wife Mrs. ANGELIN TISHA B.Tech, whose

love, support and constant patience have taught me so much about sacrifice, discipline and compromise.
At this juncture, I have a special mention about my elder brother

Dr.A.ARUL KAMALRAJ, Ph.D., and my younger brother Mr.A.ANANDHA SELVA REUBEN, MBA., without them, I couldn’t have travelled this far.

My heartful thanks to Lab Technicians, Mr. M. SUBRAMANIAN,

Mrs. S.J. MUTHUSELVI, Mrs. K. ARULMANI, Mrs. S. SHARMILA DEVI, Mrs.SUNDARAMBAL and typist Mrs.SASIKALA for their help in

technical aspects. I thank my junior Dr. D.JENIFER for her help in grossing the specimens.
I thank all my non-teaching staffs working in Department of Pathology,
Coimbatore Medical College, Coimbatore.
I would like to dedicate this book to my family’s little princess

A.BRINDHA.

I also thank my batch mates and my juniors for their love and support

Dr. A. PETER SAMIDOSS
CERTIFICATE – II

certify that this dissertation

“HISTOPATHOLOGICAL ANALYSIS OF PATIENTS

  • This
  • is
  • to
  • work
  • titled

WITH

ABRUPTIO PLACENTA IN A TERTIARY HEALTH CARE CENTRE”

of the candidate Dr. A. PETER SAMIDOSS with registration number

201613255 for the award of M.D Degree in the branch of PATHOLOGY.

I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 7% percentage of plagiarism in the dissertation.

Guide and Supervisor
CONTENTS

S.NO

1.

  • PARTICULARS
  • PAGE NO.

  • 1-4
  • INTRODUCTION

  • 2.
  • AIM OF THE STUDY

OBJECTIVES OF THE STUDY REVIEW OF LITERATURE MATERIALS AND METHODS OBSERVATION AND RESULTS DISCUSSION
5

  • 3.
  • 6

  • 4.
  • 7-37

  • 5.
  • 38-45

46-68 69-84 85-92 93-98
99-108
6. 7.

  • 8.
  • SUMMARY

  • 9.
  • CONCLUSION

10. 11.
BIBLIOGRAPHY ANNEXURES

I: Proforma and Consent Form II: Master Chart
109-110 111-112

LIST OF TABLES
S. NO.
PAGE NO.
TITLE

  • 1.
  • Age Distribution among cases and controls
  • 46

2. 3. 4. 5. 6.

  • Mean age of the study
  • 47

48 49 50 51
Correlation of clinical features among cases and control Histopathological features of Acute abruption in the study Histopathological features of chronic abruption in the study Expression of Histopathological features of acute abruption in acute abruption cases and in acute abruption with chronic features cases

7. 8.

  • Statistics - Histopathological features of acute abruption
  • 52

  • 53
  • Expression of Histopathological features of chronic

abruption in acute abruption cases and in cases of Acute abruption with chronic features

  • 9.
  • Statistics - Histopathological features of chronic abruption
  • 55

  • 58
  • 10.
  • Association of paternal smoking with cases of acute

abruption and acute abruption with chronic features

11. 12. 13.
Gender distribution of children in Placental Abruption Status of children in Placental Abruption
59 60

  • 61
  • Correlation between chorioamnionitis and preterm

deliveries among Placental Abruption
14. 15. 16.

  • Pregnancy Induced Hypertension
  • 62

63 64
Analysis of gravida between cases and control Histopathological features of acute abruption in Primi gravida and Multi gravida

  • 17.
  • Histopathological features of chronic abruption in Primi

gravida and Multi gravida
67

LIST OF CHARTS
TITLE

  • S.
  • PAGE

  • NO.
  • NO

1. 2. 3. 4. 5. 6.
Age Distribution among Cases and Controls Mean age of the study
46 47 48 49 50 51
Correlation of Clinical features among cases and control Histopathological features of Acute abruption in the study Histopathological features of chronic abruption in the study Expression of Histopathological features of acute abruption in acute abruption cases and in Acute abruption with chronic features cases

7. 8.

  • Statistics - Histopathological features of acute abruption
  • 52

  • 54
  • Expression of Histopathological features of chronic

abruption in acute abruption cases and in cases of Acute abruption with chronic features

  • 9.
  • Statistics - Histopathological features of chronic abruption
  • 56

  • 58
  • 10.
  • Association of Paternal Smoking with cases of acute

abruption and acute abruption with chronic features

11. 12. 13.
Gender distribution of children in Placental Abruption Status of children in Placental Abruption
59 60

  • 61
  • Correlation between chorioamnionitis and preterm

deliveries among placental abruption
14. 15. 16.

  • Pregnancy Induced Hypertension
  • 62

63 65
Analysis of gravida between cases and control Histopathological features of acute abruption in Primi gravida and Multi gravida

  • 17.
  • Histopathological Features of Chronic Abruption In Primi

gravida and Multi gravida
67

LIST OF COLOUR PLATES

  • S.NO
  • TITLE

1

Gross; Placental Abruption

Dilated tortuous vessels over the membranes
2

3

Gross; Placental Abruption

Maternal surface- indentation and hemorrhagic areas

Gross- Placental Abruption

Placental infarction with congestion
4

Gross – Placental Abruption

Retroplacental blood clot with vessel thrombus
5

Gross- Placental Abruption

Retroplacental hemorrhage
6

Microscopy - Placental Abruption -

funisitis
7

Microscopy - Placental Abruption

Squamous mataplasia with moderate chorioamnionitis
8

Microscopy - Placental Abruption

Squamous mataplasia with moderate chorioamnionitis
9

Microscopy - Placental Abruption

Mild chorioamnionitis
10

11 12 13

Microscopy - Placental Abruption

Moderate chorioamnionitis

Microscopy - Placental Abruption

Severe chorioamnionitis

Microscopy - Placental Abruption

moderate chorioamnionitis with haemorrhage

Microscopy - Placental Abruption

Acute deciduitis with fibrinoid necrosis
14 15 16

Microscopy - Placental Abruption

Intervillous haemorrhage

Microscopy - Placental Abruption

Intervillous and intravillous haemorrhage

Microscopy - Placental Abruption

Intervillous haemorrhage and adjacent area of acute deciduitis with haemorrhage

17

18 19 20 21 22 23 24 25 26

Microscopy - Placental Abruption

Intervillous haemorrhage and acute deciduitis

Microscopy - Placental Abruption

Intervillous haemorrhage

Microscopy - Placental Abruption

Villous haemorrhage with large area of haemorrhage

Microscopy - Placental Abruption

Moderate chorioamnionitis with adjacent area of hemorrhage

Microscopy - Placental Abruption

Increased syncytiotrophoblastic knotting

Microscopy - Placental Abruption

Increased syncytiotrophoblastic knotting

Microscopy - Placental Abruption

Increased syncytiotrophoblastic knotting with multinucleated giant cell

Microscopy - Placental Abruption

Vasculopathy(vessels with marginal hematoma)

Microscopy - Placental Abruption

Vessel with thick muscular cuffing and adjacent marginal haematoma

Microscopy - Placental Abruption

Vessel with thrombus and villous infarction

ABBREVIATIONS

: Alpha Fetoprotein : Blood Pressure

AFP BP

: Chronic Abruption-Oligohydramnios
Sequence

CAOS

: Chronic Obstructive Pulmonary Disease : Disseminated Intravascular Coagulation : Diabetes Mellitus

COPD DIC DM

: Endothelial Nitric Oxide Synthase : Human Leukocyte Antigen : Interleukin-1

eNOS HLA IL-1

: Intra Uterine Growth Retardation : Matrix Metalloproteinases : Natural Killer Cell

IUGR MMP NK CELL NO

: Nitric Oxide : Pregnancy Associated Plasma Protein A : Red Blood Cell

PAPPA-A RBC

: T- Helper 1 Lymphocytes

TH 1 LYMPHOCYTES TNF-α

: Tumor Necrosis Factor-α

INTRODUCTION

INTRODUCTION

BACKROUND OF THE STUDY

Abruptio placenta was first described by E Rigby in 1775 by publishing an essay on Uterine hemorrhage which precedes the delivery of the fetus. Placental abruption (also known as abruptio placentae) is a complication of pregnancy, wherein the placental lining has separated from the uterus of the mother prior to delivery. It is the most common pathological cause of late pregnancy bleeding. In humans, it refers to the abnormal separation after 20 weeks of gestation and prior to birth. It occurs on average in 0.5% or 1 in 200 deliveries1. In Coimbatore Medical College, the incidence of patients with placental abruption is about 1 to 2 patients per month and Intra Uterine death is 0-1 per month. Preterm deliveries associated with over half of all pregnancies complicated by placental abruption, and it leads to many adverse maternal and fetal outcomes. The etiology of placental abruption remains hypothetical but is thought to be the consequence of abnormal invasion of trophoblast leading to rupture of spiral arteries and premature separation of the placenta2.

In spite of being an unique obstetrical condition, specific diagnostic clinical criteria is not available for placental abruption. Histological evidences supportive to chronic process that often go with placental

1

abruption has led researchers to hypothesize that the condition is the end result of a chronic process which starts very early in pregnancy, and perhaps even prolonging to the time of implantation. In the presence of vaginal bleeding associated with abdominal pain, uterine tenderness or uterine contractions, placental abruption is first thought to be in the differential clinical diagnosis. Old or freshly adherent blood clots at delivery is diagnostic of abruption. Some of these placentas may have histologic features of placental abruption. Moreover, there have been cases of women diagnosed with placental abruption based on histological markers that showed a clinically unremarkable obstetrical course and outcome.

Because of these uncertainties in making the diagnosis of placental abruption, we try to find the correlation between a clinical and histologic diagnosis of placental abruption. Furthermore, we have estimated associations between acute and chronic features with placental abruption. This is to determine if clinical and pathological findings from the placenta may perhaps provide some insight as to whether placental abruption is the result of an acute event or a chronic process.

The causes of placental abruption are multifactorial, defective mechanism in early vascularization during placentation, immunologically mediated dysfunction, acute and chronic inflammatory processes might be playing significant roles in the development of placental abruption3. Many

2

maternal modifiable risk factors during pregnancy such as pregnancy induced hypertension, polyhydramnios, thrombophilia, preterm premature rupture of membranes, intrauterine infection are associated with increased risk of placental abruption.

It has been shown that trophoblast releases a factor that inhibits platelet aggregation (O'Brien et-al 1987) and it has been postulated that this factor is needed for normal placental blood flow, when it is decreased, abruption may take place. Toivonen et a l(2004) found that the low activity haplotype C-A (His 113-His139) of the microsomal epoxide hydrolase gene was less frequent in women with abruptio placenta. Tsegaselassie workalemathu et al (2013, Sep 12) conducted a study by integrating multiple genomic analytical strategies that provides opportunities for identifying novel biological pathways for exploring the underlying molecular mechanisms, for placental abruption4. In addition, genetic risk scores analyses support the promise of using genetic association studies in placental abruption risk prediction. Further efforts are needed to understand placental abruption pathological mechanisms and facilitate the development of prevention strategies5.

There are only few studies relating to abruptio placenta in primigravida patients compared to multigravida patients. Present study aims to study the histomorphological features of abruptio placenta of primigravida

3

compared to multigravida patients and to analyze the features more associated with Intra Uterine death incidence. This could enlighten us about the pathway leading to grave prognosis and fatality in such patients, thus enabling us to prevent maternal and fetal deaths in abruptio placenta.

Also though abruptio placenta is an acute event, it usually has an underlying chronic etiology like deciduitis and decidual vasculopathy. If the pathogenesis is unravelled by studying patients with placental abruption, subsequent pregnancies might be given more attention and preventive measures may be undertaken.

Thus, present study analyses the incidence, clinical features and histomorphological features of placenta in abruptio placenta patients. It also intends to compare the features of abruptio placenta with 50 control patients of normal delivery; and also differences in histomorphology of abruptio placenta in primi gravida and multi gravida patients are analysed in present study.

4

AIMS AND OBJECTIVES

AIM OF THE STUDY

To study the incidence, clinical features and histomorphological features of abruptio placenta in a tertiary health care Centre. To compare abruptio placenta with normal placenta and to analyze the differences in histomorphology of abruptio placenta in primi gravida and multi gravida patients.

5

OBJECTIVES

1. To analyse the incidence and clinical features of abruptio placenta in a tertiary health care centre.

2. To study the histopathological features of placenta in abruptio placenta and compare with normal placenta.

3. To study the morphological difference of abruptio placenta of primi gravida with that of multi gravida patients.

6

REVIEW OF LITERATURE

ANATOMY OF NORMAL PLACENTA

The only organ with defined end date and which develops in adulthood is placenta. The demand for fetal nutrition increases in its ninth week of development, where major role is played by placenta in facilitating nutrient and gas exchange between the fetal and maternal compartments 6.

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  • The Human Umbilical Cord Stem Cells Improve the Viability of OA Degenerated Chondrocytes

    The Human Umbilical Cord Stem Cells Improve the Viability of OA Degenerated Chondrocytes

    4474 MOLECULAR MEDICINE REPORTS 17: 4474-4482, 2018 The human umbilical cord stem cells improve the viability of OA degenerated chondrocytes HAO WANG1*, XU YAN2*, YUXIN JIANG3, ZHENG WANG2, YUFEI LI4 and QINGDONG SHAO2 1Teaching Center of Experimental Medicine, Shanghai Medical College, Fudan University, Shanghai 200032; 2Department of Orthopedics, 455th Hospital of PLA, Shanghai 200052; 3School of Medicine, Dalian Medical University, Dalian, Liaoning 116044; 4Department of Plastic Surgery, 455th Hospital of PLA, Shanghai 200052, P.R. China Received July 11, 2017; Accepted December 5, 2017 DOI: 10.3892/mmr.2018.8413 Abstract. Osteoarthritis (OA) affects a large number of hUC-MSCs have a predominant expression of stem cell patients; however, human umbilical cord stem cells exhibit ther- markers, while the hematopoietic and endothelial markers apeutic potential for treating OA. The aim of the present study were absent. Osteogenic, chondrogenic and adipogenic was to explore the interaction between human umbilical cord differentiation was observed in certain induction conditions. stem cells and degenerated chondrocytes, and the therapeutic hUC-MSCs improved the proliferation of OA chondrocytes potential of human umbilical cord stem cells on degenerated and downregulated the expression of inflammatory cytokines, chondrocytes. Human umbilical cord-derived mesenchymal while OA chondrocytes promoted MSCs to differentiate into stem cells (hUC-MSCs) were harvested from human umbilical chondrocytes. Taken together, the co-culture of hUC-MSCs cords,
  • Current Status and Future Prospects of Perinatal Stem Cells

    Current Status and Future Prospects of Perinatal Stem Cells

    G C A T T A C G G C A T genes Review Current Status and Future Prospects of Perinatal Stem Cells Paz de la Torre and Ana I. Flores * Grupo de Medicina Regenerativa, Instituto de Investigación Sanitaria Hospital 12 de Octubre (imas12), Avda. Cordoba s/n, 28041 Madrid, Spain; [email protected] * Correspondence: anaisabel.fl[email protected] or afl[email protected] Abstract: The placenta is a temporary organ that is discarded after birth and is one of the most promising sources of various cells and tissues for use in regenerative medicine and tissue engineering, both in experimental and clinical settings. The placenta has unique, intrinsic features because it plays many roles during gestation: it is formed by cells from two individuals (mother and fetus), contributes to the development and growth of an allogeneic fetus, and has two independent and interacting circulatory systems. Different stem and progenitor cell types can be isolated from the different perinatal tissues making them particularly interesting candidates for use in cell therapy and regenerative medicine. The primary source of perinatal stem cells is cord blood. Cord blood has been a well-known source of hematopoietic stem/progenitor cells since 1974. Biobanked cord blood has been used to treat different hematological and immunological disorders for over 30 years. Other perinatal tissues that are routinely discarded as medical waste contain non-hematopoietic cells with potential therapeutic value. Indeed, in advanced perinatal cell therapy trials, mesenchymal stromal cells are the most commonly used. Here, we review one by one the different perinatal tissues and the different perinatal stem cells isolated with their phenotypical characteristics and the preclinical uses of these cells in numerous pathologies.
  • Perinatal Derivatives: Where Do We Stand? a Roadmap of the Human Placenta and Consensus for Tissue

    Perinatal Derivatives: Where Do We Stand? a Roadmap of the Human Placenta and Consensus for Tissue

    fbioe-08-610544 December 14, 2020 Time: 16:16 # 1 REVIEW published: 17 December 2020 doi: 10.3389/fbioe.2020.610544 Perinatal Derivatives: Where Do We Stand? A Roadmap of the Human Placenta and Consensus for Tissue Edited by: Martijn van Griensven, and Cell Nomenclature Maastricht University, Netherlands Antonietta Rosa Silini1*†, Roberta Di Pietro2,3†, Ingrid Lang-Olip4†, Francesco Alviano5, Reviewed by: Asmita Banerjee6, Mariangela Basile2,3, Veronika Borutinskaite7, Günther Eissner8, Diana Farmer, 9 10 11 12 University of California System, Alexandra Gellhaus , Bernd Giebel , Yong-Can Huang , Aleksandar Janev , 12 4 13,14 United States Mateja Erdani Kreft , Nadja Kupper , Ana Clara Abadía-Molina , Aijun Wang, Enrique G. Olivares13,14,15, Assunta Pandolfi3,16, Andrea Papait1,17, Michela Pozzobon18, University of California, Davis, Carmen Ruiz-Ruiz13,14, Olga Soritau19, Sergiu Susman20,21, Dariusz Szukiewicz22, United States Adelheid Weidinger6, Susanne Wolbank6, Berthold Huppertz4† and Ornella Parolini17,23† *Correspondence: 1 Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy, 2 Department of Medicine Antonietta Rosa Silini and Ageing Sciences, G. d’Annunzio University of Chieti-Pescara, Chieti, Italy, 3 StemTeCh Group, G. d’Annunzio [email protected] Foundation, G. d’Annunzio University of Chieti-Pescara, Chieti, Italy, 4 Division of Cell Biology, Histology and Embryology, †These authors have contributed Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria,
  • A Roadmap of the Human Placenta and Consensus for Tissue and Cell

    A Roadmap of the Human Placenta and Consensus for Tissue and Cell

    fbioe-08-610544 December 14, 2020 Time: 16:16 # 1 REVIEW published: 17 December 2020 doi: 10.3389/fbioe.2020.610544 Perinatal Derivatives: Where Do We Stand? A Roadmap of the Human Placenta and Consensus for Tissue Edited by: Martijn van Griensven, and Cell Nomenclature Maastricht University, Netherlands Antonietta Rosa Silini1*†, Roberta Di Pietro2,3†, Ingrid Lang-Olip4†, Francesco Alviano5, Reviewed by: Asmita Banerjee6, Mariangela Basile2,3, Veronika Borutinskaite7, Günther Eissner8, Diana Farmer, 9 10 11 12 University of California System, Alexandra Gellhaus , Bernd Giebel , Yong-Can Huang , Aleksandar Janev , 12 4 13,14 United States Mateja Erdani Kreft , Nadja Kupper , Ana Clara Abadía-Molina , Aijun Wang, Enrique G. Olivares13,14,15, Assunta Pandolfi3,16, Andrea Papait1,17, Michela Pozzobon18, University of California, Davis, Carmen Ruiz-Ruiz13,14, Olga Soritau19, Sergiu Susman20,21, Dariusz Szukiewicz22, United States Adelheid Weidinger6, Susanne Wolbank6, Berthold Huppertz4† and Ornella Parolini17,23† *Correspondence: 1 Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy, 2 Department of Medicine Antonietta Rosa Silini and Ageing Sciences, G. d’Annunzio University of Chieti-Pescara, Chieti, Italy, 3 StemTeCh Group, G. d’Annunzio [email protected] Foundation, G. d’Annunzio University of Chieti-Pescara, Chieti, Italy, 4 Division of Cell Biology, Histology and Embryology, †These authors have contributed Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria,
  • Human Umbilical Cord Lining Cells As Novel Feeder Layer for Ex Vivo Cultivation of Limbal Epithelial Cells

    Human Umbilical Cord Lining Cells As Novel Feeder Layer for Ex Vivo Cultivation of Limbal Epithelial Cells

    Nanotechnology and Regenerative Medicine Human Umbilical Cord Lining Cells as Novel Feeder Layer for Ex Vivo Cultivation of Limbal Epithelial Cells Leonard Pek-Kiang Ang,1,2 Preeti Jain,3 Toan Thang Phan,4 and Hasan Mahmud Reza3 1Lang Eye Centre, Singapore 2Singapore National Eye Centre, Singapore 3Department of Pharmaceutical Sciences, North South University (NSU), Dhaka, Bangladesh 4Department of Surgery and Faculty of Dentistry, National University of Singapore, Singapore Correspondence: Hasan Mahmud PURPOSE. To determine the effectiveness of human umbilical cord-derived mucin-expressing Reza, Department of Pharmaceutical cord lining epithelial cells (CLEC-muc) as feeder cells in a coculture system for the cultivation Sciences, North South University, of human limbal stem cells. Bashundhara, Dhaka 1229, Bangla- desh; METHODS. Human CLEC-muc were cultured in PTTe-1 medium and treated with mitomycin C [email protected]. to arrest their growth to make the feeder layer. Single-cell suspension of limbal cells was LP-KA and HMR contributed equally prepared from corneal rim collected from the Singapore Eye Bank. Limbal cells were cultured to the work presented here and in a coculture system with CLEC-muc as well as 3T3 cells as feeder layer. We compared the should therefore be regarded as colony-forming efficiency and cell morphology of the limbal cells cultured in the two different equivalent authors. feeder layers. We also compared the expression level of several putative limbal stem cell Submitted: October 29, 2014 markers, such as HES1, ABCG2, DNP63, and BMI1, in the cultured limbal cells by Accepted: May 5, 2015 immunostaining and quantitative (q)RT-PCR.