The association between tissue non- specific alkaline phosphatase expression and differentiation ...

255 Pages·2017·8.21 MB·English

The association between tissue non-
specific alkaline phosphatase
expression and differentiation of
mesenchymal stromal cells
by
Cara-Lesley Bartlett
Dissertation presented in partial fulfilment for the degree of
Doctor of Philosophy (Internal Medicine)
at the University of Stellenbosch
Promoter: Professor William F. Ferris
Co-Promoter: Professor Nigel J. Crowther
March 2017
Stellenbosch University https://scholar.sun.ac.za
DECLARATION
By submitting this thesis electronically, I declare that the entirety of the work contained
therein is my own, original work, that I am the sole author thereof (save to the extent
explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch
University will not infringe any third party rights and that I have not previously in its entirety
or in part submitted it for obtaining any qualification.
March 2017
Copyright © 2017 Stellenbosch University All rights reserved.
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ABSTRACT
Diseases resulting from the dysregulation of adipocyte and osteoblast differentiation include
diabetes type II and osteoporosis. Both adipocytes and osteoblasts are derived from the same
progenitor cell type, known as mesenchymal stromal cells (MSCs), which may also
differentiate into cells of several mesenchymal lineages. A more detailed understanding of
the mechanisms involved in the differentiation of MSCs would provide valuable insight into
the underlying causes of, as well as facilitate the development of improved treatments for,
diseases related to MSC differentiation dysregulation.

Tissue-nonspecific alkaline phosphatase (TNAP) is highly expressed in several tissues
including bone tissue, where it has a well-established role in skeletal mineralisation. In recent
years TNAP expression has been reported in adipocytes, and has been identified as identical
to the stem cell marker, mesenchymal stem cell antigen-1 (MSCA-1). The above findings
indicate that TNAP has diverse roles, and may be one of the factors involved in determining
the differentiation pathway of MSCs.

Previous studies have found that inhibition of TNAP in the mouse preadipocyte cell line,
3T3-L1 resulted in a decrease in lipid accumulation during in vitro adipogenic differentiation,
suggesting that TNAP is involved in adipogenesis. In the present study, rat-derived primary
MSCs were isolated from bone marrow (bmMSCs) as well as subcutaneous (scADSCs) and
peri-renal visceral adipose (pvADSCs) depots, and differentiated in vitro towards either an
adipocytic or osteoblastic phenotype. The expression of TNAP was assessed in rat-derived
MSCs undergoing both adipogenic and osteogenic differentiation.

TNAP expression levels were highest in bmMSCs, followed by scADSCs and pvADSCs,
with higher alkaline phosphatase (ALP) activity observed during adipogenesis compared to
osteogenesis in all three MSC types. The addition of the reported TNAP inhibitor, levamisole
during osteogenesis prevented mineralisation in all MSC types, but had no significant effect
on lipid accumulation during adipogenesis. Other reported inhibitors were also examined;
Histidine was not successful in reducing lipid accumulation or mineralisation, while L-
homoarginine was able to significantly reduce lipid accumulation in all MSC types. The
inhibitor results were not conclusive due to possible off target effects within the cells.
Attempts to inhibit adipogenic differentiation by knockdown of TNAP expression in
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scADSCs using shRNA were not successful, as indicated by the presence of lipid droplets in
cells where TNAP-specific shRNA was present.

This study also revealed that ALP activity was localised to the membrane of intracellular
lipid droplets characteristic of adipocytes, and that the same TNAP mRNA transcript type
which is preferentially expressed in bone tissue is also preferably expressed during
adipogenic differentiation of bmMSCs and scADSCs, while expression in pvADSCs was
below detectable levels.

TNAP isoforms differ from one another due to differences in posttranslational glycosylation
pattern. Glycosylation differences were observed between bmMSCs differentiated from a
naïve state towards an adipogenic, compared to an osteogenic, phenotype. Differences were
also observed between scADSCs and bmMSCs when differentiated towards adipocytes. This
may indicate that a distinct isoform of TNAP exists in adipocytes.

In conclusion, this study confirms earlier findings on the presence of TNAP in adipocytes.
Differences in TNAP expression from MSCs isolated from different tissue depots were also
discovered. This study provides a characterisation of the role of TNAP in adipogenic
differentiation; however, the exact mechanisms remain to be elucidated.

Key words: Tissue-nonspecific alkaline phosphatase, mesenchymal stromal cell, bone-
marrow, adipocyte, osteoblast, rat, levamisole, L-homoarginine, histidine, glycosylation.

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AFRIKAANSE OPSOMMING
Die wanregulering van adiposiet en osteoblast differensiasie lei tot verskeie siektetoestande,
insluitende tipe II diabetes en osteoporosis. Beide adiposiet en osteoblast seltipes is
afstammelinge van dieselfde stamseltipe, naamlik mesenchiem stromale selle (MSSe). Meer
breedvoerige kennis van die onderliggende faktore wat bydra tot hierdie wanregulering kan
aanleiding gee tot verbeterde behandelingsmetodes vir siektes geassosieer met afwykende
MSS differensiasie.

Daar is ‘n hoë uitdrukking van weefsel nie-spesifieke alkaliese fosfatase (WNAF) in verskeie
weefseltipes, insluitende been, waar dit ‘n welbekende rol speel in skelet mineralisering het.
Daar is onlangs bevind dat WNAF in adiposiete uitgedruk word en dat dit identies is tot die
stamsel merker, mesenchiem stamsel antigeen-1. Hierdie bevindinge dui daarop dat WNAF
verskeie diverse funksies het en dat die ensiem een van die bepalende faktore mag wees wat
bydra tot differensiasie van stamselle.

Vorige navorsing het gewys dat inhibisie van WNAF in die muis pre-adiposiet sellyn, 3T3-
L1, lei tot ‘n afname in lipied akkumulasie tydens in vitro adipogeniese differensiasie. Dit
impliseer dat WNAF betrokke is by adipogenese. In die huidige studie was daar gebruik
gemaak van rot-afgeleide primêre MSSe geïsoleer uit beenmurg (bmMSSe), asook selle
verkry uit subkutaan (skAASSe) en peri-renale visserale (pvAASSe) vet depots. Hierdie selle
was gedifferensieer, in vitro, in ‘n adiposiet of osteoblast fenotipe. Die uitdrukking van
WNAF was bepaal in rot-afgeleide MSSe wat gestimuleer was om of adipogeniese of
osteogeniese differensiasie te ondergaan.

Vlakke van WNAF uitdrukking was die hoogste in bmMSSe gevolg deur skAASSe en
pvAASSe. Daar was meer alkaliese fosfatase aktiwiteit tydens adipogenese in al drie MSS
tipes. Verder het die WNAF inhibitor, levamisool, mineralisering tydens osteogenese
voorkom in al drie MSS tipes. Die inhibitor het egter geen beduidende effek gehad op lipied
akkumulasie tydens adipogenese nie.

Die effek van twee ander moontlike WNAF inhibitore, histidien en L-homoarginien, was ook
ondersoek. Histidien het nie lipied akkumulasie of mineralisering onderdruk nie.
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Inteenstelling hiermee het L-homoarginien lipied akkumulasie beduidend in al drie MSS tipes
verminder. Resultate verkry met inhibitore was nie beslissend nie. Dit mag wees as gevolg
van moontlike buite-teiken effekte van die inhibitore in hierdie selle. Dus was daar gepoog
om adiposiet differensiasie te inhibeer deur die afklop van WNAF uitdrukking in skAASSe.
Dit was bewerkstellig deur gebruik te maak van klein haarnaald RNAs (khRNAs). Selle het
egter steeds lipied druppels vertoon in die teenwoordigheid van WNAF spesifieke khRNAs.

Eksperimente in hierdie tesis het ook onthul dat WNAF aktiwiteit gelokaliseer is in die
membraan van intrasellulêre lipied druppels. Verder is daar ook gedemonstreer dat dieselfde
WNAF boodskapper RNA-transkripsie tipe wat met voorkeur in beenweefsel uitgedruk word,
ook met voorkeur tydens adipogeniese differensiasie uitgedruk word in bmMMSe en
skAASSe. Die uitdrukking van hierdie transkripsie tipe was nie in pvAASSe gevind nie.

Verskille in post-translasie glikosilasie gee aanleiding to verkillende isoforme van WNAF.
Glikosilasie-verskille was gesien in MSSe differensiasie in ‘n adipogeniese fenotipe in
vergelyking met differensiasie in ‘n osteogeniese fenotipe. So ook was daar verkille gemerk
tussen skAASSe en bmMSSe wat gedifferensieer was in ‘n adiposiet fenotipe. Dit mag
aandui dat ‘n eiesoortige WNAF isoform in adiposiete voorkom.

Bogenoemde saamgevat, die huidige studie bevestig vroeëre bevindinge dat WNAF
teenwoordig is in adiposiete. Verskille is gewys in die uitdrukking van WNAF in MSSe
geïsoleer uit versillende weefsel depots. Hierdie studie voorsien ‘n karakterisering van die rol
wat WNAF speel in adipogeniese differensiasie, maar die presiese funksie van WNAF is
egter nog ontwykend.

Sleutel woorde: weefsel nie-spesifieke alkaliese fosfatase, mesenchiem stromale selle,
beenmurg, vetsel, rot, levamisool, L-homoarginien, histidien, glikosilasie.

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ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to the following people and organisations:

The MRC, Stellenbosch University FMHS and the Harry Crossley foundation for funding.

My supervisor Prof. William Ferris and my co-supervisor Prof. Nigel Crowther, for their
expert guidance and supervision throughout the duration of this project.

The late Prof. Stephen Hough, for his valued advice and expertise.

Dr. Hanél Sadie-van Gijsen for her technical advice and training.

Dr. Mari Van De Vyver for assistance with data processing and statistics.

To my colleagues and dear friends, the “coffee club”: Dr. Ellen Andrag, Alex Jacobs and
Martine van den Heever, for the years of friendship, advice, shared expertise, and the many
fruitful scientific discussions over the years.

Dr. Ellen Andrag, for help with translation of the abstract into Afrikaans.

Finally, to my parents, Alan and Gail Bartlett, for their endless love, support and
encouragement throughout my university career.

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TABLE OF CONTENTS
ABSTRACT ............................................................................................................................... ii
AFRIKAANSE OPSOMMING ................................................................................................. iv
ACKNOWLEDGEMENTS ....................................................................................................... vi
LIST OF ABBREVIATIONS .................................................................................................. xii
LIST OF FIGURES ................................................................................................................. xvi
LIST OF TABLES ................................................................................................................... xxi

CHAPTER 1 .............................................................................................................................. 1
Alkaline phosphatase (ALP) and the osteogenic and adipogenic differentiation
of mesenchymal stromal cells (MSCs)..................................................................................... 1
1.1 Introduction ........................................................................................................................... 2
1.2 Literature Review.................................................................................................................. 4
1.2.1 Alkaline phosphatase background ..................................................................................... 4
1.2.2 Biochemistry of ALPs........................................................................................................ 5
1.2.3 Alkaline phosphatase structure and function ..................................................................... 5
1.2.4 Mammalian ALP isozymes ................................................................................................ 9
1.2.4.1 Tissue-specific ALP isozymes ...................................................................................... 11
1.2.4.2 Tissue-nonspecific ALP (TNAP) .................................................................................. 13
1.2.5 Genetics of TNAP ............................................................................................................ 19
1.2.6 Genetic mutations of TNAP – hypophosphatasia ............................................................ 21
1.2.7 Mesenchymal stromal cells (MSCs) ................................................................................ 22
1.2.7.1 Bone marrow-derived mesenchymal stromal cells (bmMSCs) ..................................... 25
1.2.7.2 Adipose-derived mesenchymal stromal cells (ADSCs) ................................................ 26
1.2.8 MSC differentiation – osteoblastogenesis and adipogenesis ........................................... 27
1.2.8.1 Osteoblastogenesis ........................................................................................................ 28
1.2.8.2 Adipogenesis ................................................................................................................. 34
1.2.10 Bone morphogenetic proteins ........................................................................................ 35
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1.2.11 Other factors influencing MSC differentiation .............................................................. 35

CHAPTER 2 ............................................................................................................................ 39
Materials and Methods ........................................................................................................... 39
2.1 Materials ............................................................................................................................. 40
2.2 Methods............................................................................................................................... 40
2.2.1 Isolation of rat mesenchymal stromal cells from subcutaneous and
visceral adipose tissue depots. .................................................................................................. 40
2.2.2 Isolation of rat mesenchymal stromal cells from bone marrow. ...................................... 42
2.2.3 Cell culture maintenance and Passage ............................................................................. 43
2.2.4 Alkaline phosphatase (ALP) extraction and activity ....................................................... 43
2.2.4.1 Cell lysates preparation for alkaline phosphatase (ALP) activity assay ........................ 44
2.2.4.2 Alkaline phosphatase extraction .................................................................................... 44
2.2.4.3 Alkaline phosphatase activity assay .............................................................................. 45
2.2.4.4 Protein concentration determination ............................................................................. 47
2.2.5 Adipogenic differentiation ............................................................................................... 47
2.2.6 Oil Red-O staining ........................................................................................................... 48
2.2.7 Osteogenic differentiation ................................................................................................ 48
2.2.8 Alizarin red staining for mineralisation ........................................................................... 49
2.2.9 Quantification of lipid accumulation and mineralisation by image analysis ................... 49
2.2.10 Surface marker expression of MSCs by FACS analysis ................................................ 50
2.2.11 5-bromo-2’-deoxyuridine (BrdU) assay for cellular proliferation ................................. 51
2.2.12 Nucleic acid methods ..................................................................................................... 52
2.2.12.1 Total RNA isolation .................................................................................................... 52
2.2.12.2 cDNA synthesis ........................................................................................................... 53
2.2.12.3 Conventional PCR ....................................................................................................... 54
2.2.12.4 Quantitative real-time PCR (RT-qPCR) ...................................................................... 55
2.2.13 Fluorescence microscopy – optimization of perilipin A staining in
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subcutaneous and visceral adipose derived stromal cells. ........................................................ 57
2.2.13.1 Immuno-fluorescent Perilipin A staining .................................................................... 57
2.2.13.2 Alkaline phosphatase activity detection using a fluorescent probe. ............................ 58
2.2.14 Knockdown of tissue-nonspecific alkaline phosphatase (TNAP) gene (Alp1)
using the MISSION® viral transduction system (Sigma) ......................................................... 58
2.2.15 Glycosylation analysis of TNAP.................................................................................... 60
2.2.16 Statistical analysis .......................................................................................................... 61

CHAPTER 3 ............................................................................................................................ 62
Characterisation of mesenchymal stromal cells (MSCs) from adipose and
bone marrow depots. .............................................................................................................. 62
3.1 Introduction ......................................................................................................................... 63
3.2 Results ................................................................................................................................. 66
3.2.1 Mesenchymal stem cell (MSC) morphology ................................................................... 66
3.2.2 Mesenchymal stem cell (MSC) surface marker expression ............................................. 69
3.2.3 Proliferation ..................................................................................................................... 72
3.2.4 Differentiation .................................................................................................................. 74
3.2.4.1 Adipogenic differentiation ............................................................................................ 74
3.2.4.2 Osteogenic differentiation ............................................................................................. 78
3.3 Discussion ........................................................................................................................... 83

CHAPTER 4 ............................................................................................................................ 90
Effects of TNAP inhibitors on lipid accumulation, mineralisation and ALP
activity levels of MSCs undergoing adipogenic or osteogenic induction. .......................... 90
4.1 Introduction ......................................................................................................................... 91
4.2 Results ................................................................................................................................. 93
4.2.1 Selection of TNAP inhibitor concentrations .................................................................... 96
4.2.2 Evaluation of the effect TNAP inhibitors on bmMSC osteogenesis and
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