IN VIVO EX VIVO OSTEOGENESIS OF HUMAN EMBRYONIC STEM CELLS

Aim This study was aimed at comparing the in vivo osteogenic differentiation potential of human embryonic stem cells hESC and human somatic osteoblast cell line.. Both hESCs and hFOB c

IN VIVO AND EX VIVO OSTEOGENESIS OF HUMAN

EMBRYONIC STEM CELLS

DR SUBAKUMAR LAKSHMI

(B.D.S., Dr MGR Medical University, India)

A THESIS SUBMITTED FOR THE DEGREE OF

MASTER OF SCIENCE

DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY

FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE

2009

ACKNOWLEDGEMENTS

I am extremely lucky to have met a number of wonderful people during the course of my masters’ program at Faculty of Dentistry, NUS My utmost gratitude goes to my supervisor, Associate Professor Yeo Jin Fei, for giving me the opportunity to pursue this research program Without his warm support and encouragement, I would not be where I am today

I am deeply grateful to my co-supervisor Associate Professor Cao Tong for his generous support and unfailing guidance throughout this work It is no exaggeration to say that without his support this thesis would not have happened

I would like to specially thank Dr.Yang Zheng for spreading her enthusiasm and extending her guidance during the many challenging days I am indebted to her for having critically reviewed my thesis and helped me improve my skills in scientific writing

I would like to thank Dr Ge Zigang and Dr Alexis Heng Boon Chin for their support I would like to extend my gratitude to Ms Han Tok Lin for patiently training

me in histological work My heartfelt thanks go to my colleagues Dr Vinoth Kumar,

Dr Liu Hua, Mr Toh Wei Seong, Mr Lu Kai, Ms Fu Xin, Ms Sui Lin, and Mr Li MingMing for their valuable help and making my stay in NUS memorable

TABLE OF CONTENTS

1 LITERATURE REVIEW 2

1.1 Bone 2

1.1.1 Bone composition 1 2

1.1.2 Bone structure 2 4

1.1.3 Bone types 5

1.1.4 Bone formation and remodelling 1 5

1.2 Clinical repair of bone defects 7

1.3 Cell based tissue engineering 10

1.3.1 Scaffolds 10

1.3.2 Materials used as scaffolds 10

1.3.3 Seeding of cells into scaffolds 12

1.3.4 Cell source for cell-based tissue engineering for bone regeneration 12

1.4 Stem cells in bone repair 13

1.5 Human Embryonic stem cells 15

1.5.1 Derivation of HES cells 15

1.5.2 Expansion and maintenance of pluripotency of HES cells 16

1.5.3 Characterization of hES cells 18

1.6 Differentiation of hES cells into osteogenic lineage 18

1.7 In vivo bone formation using Stem Cells 20

2 MATERIAL & METHODS 23

2.1 Culture and maintenance of Human Embryonic Stem Cells 23

2.2 Initiation of osteogenic differentiation in hESCs 24

2.3 Human fetal osteoblasts and their culture conditions 24

2.4 Methods for assessment of in vitro osteogenic differentiation 25

2.4.1 RNA extraction and c-DNA synthesis 25

2.4.2 Conventional Polymerase Chain Reaction 25

2.4.3 Detection of calcium deposition by alizarin red staining 26

2.4.4 Hoechst DNA quantification assay 26

2.5 Embedment of H1 & hFOB cell in Extracel TM 27

2.5.1 Assessment of cell viability in Extracel TM 27

2.6 In vivo experiments 28

2.6.1 Culture of H1 cells in Matrigel 28

2.6.2 Pre-differentiation of cells in scaffolds prior to in vivo implantation 29

2.6.3 Labeling of differentiating cell with CFDA-SE 29

2.6.4 In vivo grouping 30

2.6.5 Surgical procedure 30

2.6.6 Tissue Processing 31

2.7 Histology Staining 31

2.7.1 H & E staining 31

2.7.2 von Kossa staining 31

2.7.3 Immuno-Fluoresence Staining 32

3 RESULTS 34

3.1 Characterisation of hESCs 34

3.2 In vitro results - Confirmation of osteodifferentiation in hESCs and hFOB - 2D culture system 35

3.2.1 Alizarin red staining 35

3.2.2 Hoechst DNA Quantification Assay 37

3.2.3 PCR Results 37

3.2.4 Cell viability in 3D culture system - Laser Scanning Confocal Microscopy 38

3.3 In vivo results – Histology 38

3.3.1 Samples removed after 2 weeks in vivo 40

3.3.2 Samples removed after 4 weeks in vivo 42

3.3.3 Samples removed after 7 weeks in vivo 44

4 DISCUSSION 53

5 CONCLUSION 59

5.1 Limitations and future direction 60

6 APPENDIX 62

7 REFERENCES 64

Aim

This study was aimed at comparing the in vivo osteogenic differentiation

potential of human embryonic stem cells (hESC) and human somatic osteoblast cell line

Method

HESCs were propagated on mouse embryonic feeder cells They were shown

to be pluripotent by expression oct4, sox2 and nanog molecular markers Human fetal osteoblasts (hFOB) cell lines were cultured in DMEM media without phenol red Osteogenic differentiation was initiated by supplementing the culture medium with β-

glycerophosphate, ascorbic acid, dexamethasone and vitamin D3 After 21 days of in vitro culture, osteogenesis in both cell types was confirmed by expression of

osteocalcin and bone sialprotein molecular markers and calcium deposition by alizarin red staining

For in vivo assessment of osteogenesis, hESCs were propagated on feeder

free culture system for 2 weeks Both hESCs and hFOB cells were then seeded into PLGA scaffolds with cell carrier, Extracel, and left in culture media containing the

same osteogenic supplements used for in vitro osteogenic differentiation Before going in vivo the cells were stained with CFDA, a cell tracing reagent After 14 days

of in vitro differentiation, the cell-scaffold constructs were placed subcutaneous into

the dorsum of the mice Sacrifices were made at the end of 2nd, 4thand 7th weeks after implantation The removed samples were processed and stained to assess the capacity of both hESCs and hFOB to form mineralized tissue The tissue sections

were stained with H&E, von Kossa followed by immunostaining for osteonectin and alkaline phosphatase to confirm osteogenic differentiation

Results

In hESC differentiated samples harvested after 7 weeks of implantation, discrete areas of mineralized tissue could be observed within the scaffolds This was confirmed by von Kossa staining, H&E staining and immunostaining with osteonectin and alkaline phosphatase (ALP) There was no evidence of teratoma formation In hFOB samples harvested 7 weeks after implantation, immunostaining with osteonectin and ALP confirmed the progression to osteogenic differentiation within the scaffolds However, the samples did not stain positive for von Kossa staining A possible explanation for this could be that the hFOB cells were at an early stage in differentiation

In samples harvested after 4 weeks of implantation, the scaffolds were found

to be empty and devoid of the implanted cells This was observed in samples of both cell types

Mineralization could not be detected in samples of both cell types harvested after 2 weeks of implantation

Conclusion

This study demonstrates the possibility of generating osteoblasts from direct differentiation of hESCs under our osteogenic conditions These osteoblasts when implanted subcutaneous into SCID mice, where able to form mineralized tissue A

comparison of in vivo osteogenesis in hFOB and hESCs was done This comparison helped us to confirm the efficacy of in vivo osteogenesis in hESC

LIST OF FIGURES

Figure 1 (a) human ES colonies on embryonic fibroblast feeder layer (b) RT-PCR analysis

of Oct4, Nanog and Sox2 in undifferentiated hESCs 34

Figure 2 Alizarin red staining of mineralized nodules in H1 derived osteoblasts and hFOB

on day 7 and day 21 of in vitro osteogenic culture 36

Figure 3 RT-PCR analysis of osteocalcin expressions in H1 derived osteoblasts and hFOB

on day 7 and day21 of osteogenic culture 39

Figure 4 FDA staining shows viable H1 cells (green), PI staining shows dead H1 cells (red)

39

Figure 5 Cell-scaffold construct extraction after 7-weeks in vivo 39

Figure 6 At 2-week time point: H&E staining and von Kossa staining of sections showing

scaffold and cells (e) hFOB - higher magnification showing scaffold (arrow) 41

Figure 7 At 4-week time point: H&E staining and von Kossa staining of sections showing

scaffolds devoid of cells 43

Figure 8 At 7 week time-point: (a) H&E staining showing newly formed tissue within the

scaffold (arrow showing scaffold remains) (b) von Kossa staining and counterstaining with nuclear fast red showing deposition of mineralized tissue in the area marked and shown in higher magnification: x20 Sites of mineralization indicated by arrow in fig (d ), (c) H&E staining showing newly formed tissue (arrow showing blood vessel infiltration into newly formed tissue) 45

Figure 9 7 week time-point (b) Immunostaining for osteonectin (arrow) (d) Immunostaining

for bone specific ALP (arrow) (a) (c) nuclear counterstaining with DAPI (blue) 46

Figure 10 Combination picture showing (a) H1generated osteoblasts taking up dapi,

osteonection & CFDA stains (arrow) (b) H1 generated osteoblasts taking up dapi, ALP & CFDA stains (arrow) (c) & (d) CFDA-SE staining showing the implanted cells in tissue

sections 47

Figure 11 At 7 week time-point: (a) H&E staining (c) H & E showing newly formed tissue

(arrow showing blood vessel), (b) von Kossa staining did not show the presence of

mineralised tissue 49

Figure 12 At 7 week time-point: (a) (c) nuclear staining with DAPI (b) Immunostaining with

bone specific ALP (arrow showing the cells positive for ALP stain) (d) Immunostaining with osteonectin (arrow showing cells positive for osteonectin staining) 50

Figure 13 Combination pictures (a) hFOB taking up dapi, osteonection & CFDA stains

(arrow) (b) hFOB taking up dapi, ALP & CFDA stains (arrow) (c) & (d) CFDA-SE staining showing implanted cells 51

CHAPTER 1 LITERATURE REVIEW

1 LITERATURE REVIEW

1.1 Bone

Bone is a mineralized connective tissue that acts as an internal scaffold for the body Arranged both inside and around the bone are softer structures such as the vital organs, muscles, cartilages, nerves, blood vessels, adipose tissue and skin Bone also serves as a reservoir for minerals such as calcium, phosphate and magnesium

Its unique cellular content and structural organization helps it to meet its

biochemical and biomechanical demands in vivo

1.1.1 Bone composition 1

Bone consists by weight of about 67% inorganic and 33% organic substances The organic substances include both collagen and noncollagenous proteins such as proteoglycans, bone sialoprotein, osteocalcin, osteopontin and osteonectin This organic matrix is permeated by hydroxyapatite crystals which constitute the inorganic portion of the bone These crystals are responsible for a physiological mechanism called mineralization that happens in bone

The cellular components responsible for maintenance of the bone architecture include the osteogenic cells and osteoclasts Osteogenic cells which form and maintain bone are represented by osteoprogenitors, preosteoblast, osteoblasts, osteocytes and bone lining cells constituting the different maturation stages of osteogenic cells Osteoclasts are responsible for physiological bone resorption and remodeling of bone tissue

Osteoblasts are cells derived from the mesoderm Under the microscope, these mononucleated cells are distinct with their presence of extensive endoplasmic reticulum and numerous free ribosomes in the cytoplasm suggestive of their strong role in protein synthesis Osteoblasts are cells specialized to perform two important functions, extracellular matrix formation and mineralization Osteoblasts synthesize both the collagenous and non collagenous bone matrix proteins This uncalcified bone matrix called the osteoid acts as a scaffold for deposition of the apatite crystals Some osteoblasts, during their process of bone formation get trapped within their own matrix forming osteocytes Generally, woven bone has more osteocytes than the lamellar bone These osteocytes after their formation become reduced in size The space in the bone matrix occupied by an osteocyte is referred to as osteocytic lacuna Extensions of these lacunae form cannaliculi and house the osteocytic processes Through these channels and processes, the osteocytes maintain contact with adjacent osteocytes, osteoblasts and the bone lining cells This places the osteocytes in a very ideal position to sense the biochemical and mechanical environment around them and transduce signals to other cells involved

in maintenance of bone integrity and vitality

When bone is no longer forming, osteoblasts flatten and extend along the bone surfaces These cells are called bone lining cells

Osteoclasts are bone resorbing cells derived from the hematopoietic lineage Typically, these cells are found in hollowed out depressions called the Howships lacunae Under a scanning electron microscope these lacunae appear as shallow troughs with irregular shapes, reflecting the activity and mobility of osteoclasts during active bone resorption

Osteoclasts are multinucleated cells with cell membrane thrown into numerous folds to form ruffled borders At the periphery of the border, the plasma membrane is apposed closely to the bone surface This clear zone not only attaches the osteoclasts to the mineralized surface but also isolates a microenvironment between them and the bone surface

Another feature of osteoclasts is a proton pump associated with the ruffled border that pumps hydrogen ions into the sealed compartment The sequence of resorption is as follows:

1 Osteoclasts attach to the mineralized surface of the bone

2 A sealed microenvironment is created through the action of proton pump

which demineralizes bone and exposes the organic matrix

3 The exposed bone matrix is degraded by enzymes such as acid phosphatase and cathepsin B

4 Endocytosis of the degraded organic matrix happens at the ruffled borders

5 These degraded products are released along the membrane opposite the ruffled border

1.1.2 Bone structure 2

A section of a bone would reveal a dense sheet of outer compact bone and a central, medullary cavity filled with red or yellow bone marrow that is interrupted by a network of bone trabeculae This network of bone trabaculae is called the cancellous

or spongy bone

Adult bone whether compact or spongy consists of microscopic layers or lamellae Three distant types of layering can be seen under the microscope –

circumferential lamellae, concentric lamellae with the Haversian canal and the interstitial lamellae

Surrounding the outer aspect of the compact bone is a connective tissue membrane called the periosteum The outer layer of periosteum consists of fibrous connective tissue The inner layer of periosteum consists of bone cells, their precursors and a rich vascular supply Calcification prevents diffusion of nutrients into all the bone cells Periosteum thus provides these cells with direct vascular supply

1.1.3 Bone types

Many types of classifications exist for bone Based on their shape, bone can

be classified as long bones (eg: femur, tibia, humerus, fibula), short bones (eg: carpals, metacarpals, tarsals, metatarsals), flat bones (eg: frontal, parietal, scapula) and irregular bones (eg: maxilla, sphenoid) Bone can also be broadly classified as woven bone and lamellar bone Woven bone is highly cellular, formed in response to growth or injury and rich in bone sialoprotein Lamellar bone is the mature bone which has collagen fibers arranged in lamellae and contains large quantities of osteocalcin Bones can also classified into dense outer cortical bone and inner cancellous or spongy bone

1.1.4 Bone formation and remodelling 1

Throughout embryogenesis bone development happens by two distinct processes Intramembraneous ossification occurs in the bones of cranial vault, maxilla, and body of the mandible and the midshaft of long bones In this type of ossification, bone develops directly within the soft connective tissue First,

mesenchymal cells proliferate and condense as a membrane in the area of future bone Vascularity at these sites increases Osteoblasts differentiate from the mesenchymal cells and begin to produce bone matrix The newly formed bone is termed woven bone From early fetal development to full expression of the adult skeleton a continual slow transition occurs from woven bone to lamellar bone

Endochondral ossification occurs at extremities of long bones, vertebrae, ribs and at the articular extremities of mandible and the base of the skull First there is condensation of mesenchymal cells Chondrocytes differentiate from these condensed mesenchymal cells to form cartilage The cartilage then undergoes a degenerative process with chondrocyte hypertrophy, programmed cell death and matrix calcification Blood vessels penetrate bringing osteoprogenitor cells to the region which after differentiating to osteoblast, produced bone matrix overlaying the cartilaginous matrix remnants

Establishment of the overall size and shape of the bone extends from intrauterine stage to preadult period of human growth During this phase bone is rapidly formed in the periosteal surface and destroyed along the endosteal surface within the compact bone The adult skeleton also undergoes remodeling In a healthy individual the amount of bone lost is balanced by the amount of bone formed

However, in case of certain diseases and with age, bone resorption exceeds formation resulting in overall loss of bone Also defects that arise due to trauma, tumors or abnormal skeletal development cannot be addressed by the normal bone remodeling processes

1 Ten Cate’s oral histology: Development, structure and function

2 The anatomical basis of dentistry, Bernard Liebgott

1.2 Clinical repair of bone defects

Orthopedists, oral surgeons and scientists have always been looking for ways to stimulate fracture repair, heal non-union or restore lost segments of bone Currently, skeletal defects are largely addressed in clinics by autogenous bone grafts, allogeneic bone grafts or by bone graft substitutes which include demineralized bone matrix, ceramics, graft composites and bone morphogenetic protein

The biology of these grafts varies and may provide one or more of these several essential components: (1) Osteogenic cells (2) Osteoconductive matrix (3) Osteoinductive proteins (Finkemeier, 2002).

Autografts are derived from the patient’s own bone These grafts have osteogenic, osteoinductive and osteoconductive properties Common harvest sites for these grafts include iliac crest, tibia, fibula and radial bone Advantages of autografts include their excellent success rate, low risk of transmission of diseases and histo-compatibility However the disadvantages include the limitation in availability of graft material and the potential donor site morbidity Tang et al reported that of the thirty nine patients who had a free fibular graft harvested for treatment of avascular necrosis of femoral head, 42% had a subjective sense of instability and 37% had a subjective sense of weakness in the lower extremities potentially due to muscle stripping during fibula harvest

Another source of autogenous graft material is the autogenous bone marrow The injection of bone marrow provides a graft that is osteogenic (bone marrow stem cells) and osteoinductive (cytokines and growth factors secreted by the marrow cells) Bone marrow can be aspirated from the posterior iliac crest and injected into

the defect The tendency of the injected materials to be washed away from the defect site is a potential problem in this technique Demineralised bone matrix which

is both an osteoconductive and osteoinductive material is used as carrier for autogenous bone marrow Injection of autogeneous bone marrow with or without carriers has been used to treat several bone defects in clinics

With the development of immune suppressant, allogenic bone grafts are an alternate option and are available in various preparations Demineralized bone matrix, cancellous chips, corticocancellous and cortical grafts, osteochondral and whole bone segments are some of them Cortical, cancellous, osteochondral and the whole bone segments that are available are osteoconductive and provide immediate mechanical support These are typically non vital bones harvested from cadavers and processed for use in clinics However, most allografts possess the risk of disease transmission

Demineralised bone matrix (DBM) is prepared by a standardized process in which allogeneic bone is crushed into small particles and demineralized in HCL DBM preparations are available as gels, puttys or strips for clinical use These grafts offer both as osteoconductive and osteoinductive properties However they do not offer structural support Another disadvantage of demineralized bone matrix is that different batches may have different potencies because of the wide variety of donors used (Finkemeier, 2002)

Bone graft substitutes are commercially available alternatives to auto and allogeneic bone grafts which possess many of the bone forming properties as human bone Ceramics, ceramics phosphates and bioactive glasses are a few examples of these substitutes available for clinical use Calcium phosphate ceramics are

osteoconductive and are used to fill bone defects But these crystals by themselves are brittle and have poor tensile strength Hence they should be placed in intact bone

or rigid stabilized bone in order to protect the ceramic from shear stress and they should be tightly packed into adjacent host bone to maximize in-growth (Bucholz et

al, 1987) Another bone graft substitute currently in clinical use is calcium collagen graft material This graft also does not provide structural support but is used as a bone graft expander to augment fracture repair

Bone morphogenetic proteins (BMP) are proteins naturally produced in the body to regulate bone formation and healing. Recombinant human bone morphogenetic protein (rhBMP) is an osteoinductive material currently available and FDA approved for treatment in certain kinds of bone defects Combinations BMP with gelatin foam, collagen, or calcium phosphate pastes is said to increase its retention in the defect site Clinical studies have shown higher BMP doses are needed for osteoinductivity in humans (Termaat et al, 2005) Also the production and

purification of BMP makes this an expensive treatment

To summarize, graft rejection, shortage in graft material, insufficient biocompatibility, lack of strength of the graft material and absence of cells with reparative potential to fill in large defects are the major disadvantages faced by most

of these treatment methods

address this growing problem

1.3 Cell based tissue engineering

Cell based tissue engineering deals with the use of biodegradable materials seeded with living cells to regenerate the form and function of a damaged or diseased tissue in a human body Tissue engineering could be seen as a tool for regenerative medicine The concept of tissue engineering involves cells on

biodegradable scaffolds to generate functionally active tissues for in vivo

applications Investigations into the cell types available for this purpose, the scaffolds used and their combined role in bone formation are the need of the hour

1.3.1 Scaffolds

The ideal scaffolds are 3-dimensional, porous, osteoconductive biomaterials that have the following functions to perform: (1) promote cell adhesion and extracellular matrix deposition (2) permit the transport of nutrients, gases (3)

biodegrade at a controlled rate (4) provoke minimal inflammation or toxicity in vivo

While considering scaffolds for bone regeneration, appropriate mechanical properties and design have to be taken into consideration (Principles of Tissue Engineering, Lanza, Langer and Vacanti)

1.3.2 Materials used as scaffolds

Natural Polymers

Collagen scaffolds have been reported to heal tibial bone defects in rats (Rocha et al, 2002) Collagen being the most commonly available protein in the body has the following advantages - excellent biocompatibility, biodegradability and promoting cell adhesion But the disadvantages have been its low mechanical strength Scaffolds made from hyaluronic acid have been used for osteogenic

differentiation of murine pluripotent cells (Kim and Valentini, 2002) But, like collagen they also fail to meet the mechanical demands

Ceramics

Bioceramics such as hydroxyapatite and tricalcium phosphate, because of their similarities in composition with the inorganic portion of bone, have been used as scaffolding materials for bone regeneration However, the major limitations in their use include their brittleness and difficulty in processing

Synthetic Polymers

Biodegradable homopolymers and copolymers of lactic and glycolic acid have also been frequently used as scaffold materials Many types of cells have been shown to attach and grow on these materials Neonatal rat osteoblasts have been shown to attach to PLA (poly lactic acid), PGA (poly glycolic acid), PLGA (poly lactic

co glycolic acid) substrates and synthesize collagen in culture (Ishaug et al, 1994) PCL (polycaprolactone) -HA (hydroxyapatite) scaffolds were used to tissue engineer bone in a study conducted by Yu et al in 2008

These synthetic polymers have for long been used in clinics as degradable suture materials PLGA scaffolds are biocompatible, undergo controlled biodegradability and have good mechanical properties In addition, reports have demonstrated that PLGA scaffolds support proliferation and differentiation of osteoprogenitor cells (Yang et al, 2001) Lactic acid and glycolic acid are end products of their biodegradation that are eventually metabolized by the body In addition, they have a high three dimensional design flexibility and can be fabricated into scaffolds with different size and structure

The PLGA scaffolds used in this study are porous with a pore size of about 50

µm and channel size of about 2.4 mm

1.3.3 Seeding of cells into scaffolds

Various gels have been used to carry cells into scaffolds An ideal cell carrier would be one which supports homogeneous and high cell-seeding efficiency as well

as proliferation/differentiation While considering gels for this purpose the following requirements have to be met: (1) close to neutral pH (2) quick setting time (3) stable

in the culture media during the in vitro culture (4) biodegradation approximates that

of scaffolds (5) minimal toxic residue

Extracel TM used as a cell carrier in this study is a hydrogel that is made up of three biocompatible components: thiol-modified hyaluronon, thiol-modified gelatin and a thiol-reactive crosslinker; polyethylene glycol diacrylate Collagen and hyaluronic acid are of bovine origin

1.3.4 Cell source for cell-based tissue engineering for bone regeneration

Cellular components used in tissue engineering encompass viable cells of autologous, allogeneic or animal origin While using viable cells, care is needed to control introduction of infectious diseases, cross-contamination from donors or introduction of infectious agents from materials used to process cells Also, consequences of immune and inflammatory responses after the implantation of the cell- scaffold construct need to be considered

Different types of cells have been used for the reconstruction of bone tissue Periosteal cells, skeletal muscle cells (Deasy et al, 2001), cells derived directly from

bone, as well as cells transduced with bone morphogenetic protein genes (Lee JY et

al, 2001) have been used

Xiao et al in 2003 concluded that cells from human alveolar bone can retain their osteogenic properties in a three-dimensional collagen scaffold and subsequently synthesize a bone matrix, which after implantation in SCID mice can induce new bone formation in critical-size calvarial bone defects Muschler and Midura, (Hutmacher and Sittinger, 2003) based on their mathematical model, calculated that 70 million osteoblasts are needed to regenerate 1cm3 of bone But, there are limitations in expanding differentiated osteoblasts to such high numbers

Vacanti et al in the year 2001 (Hutmacher and Sittinger, 2003) reported a clinical case in which periosteal cells were used in bone tissue engineering A biopsy taken after 10 months revealed that the cell-scaffold construct was vascularized and well integrated into the tissues However, only 5% of the constructs showed newly formed lamellar bone and endochondral tissue

A clinical trial conducted by the University of Freiburg in 2003 (Hutmacher and Sittinger, 2003) resulted in commercial availability of bone grafts using autologous periosteal cells for clinical applications (BioSeed B; BioTissue Technologies, Freiburg, Germany) However, autologous procedures are time consuming due to the prolonged ex vivo cell culture

1.4 Stem cells in bone repair

Stem cells are the fundamental source of tissue for any organism They provide the body with cells that are needed both for growth as well as repair Stem cells are found both in a developing embryo as well as in an adult body

Stem cells can be broadly classified based on the variability of derivatives they give rise to Unipotent stem cells can generate only one mature cell type, multipotent stem cells give rise to two or more differentiated cell types and pluripotent stem cells that can give rise to representative of all three germ layers

Two important sources of stem cells available for regenerative therapy include (1) the adult stem cells from the adult tissue (autologous or allogenic) and from the bone marrow (mesenchymal stem cells, hematopoietic stem cells) and (2) embryonic stem and embryonic germ cells derived from discarded human embryos and germ line stem cells

Adult stem cells can be derived from various parts of the body including bone marrow, blood, deciduous teeth, pulp tissue and muscle These lineage-restricted stem cells have been isolated from both fetal and adult tissues for application in regenerative medicine Although, these cells have a high capacity to self renew in culture, their ability to expand is less than that for embryonic stem cells The self renewal and the proliferative capacity of these stem cells also decrease with age (Pittinger et al, 1999; Murphy et al, 2002; Heng et al 2004) Recent studies have shown that MSCs derived from different tissue origins have different potential in lineage differentiation (Im et al, 2005; Chen et al 2006; Kern et al, 2006) In general MSCs are a heterogeneous collection of progenitor cells with varying degrees of replicative potential Thus purification is required for its use in lineage specific differentiation

In comparison, embryonic stem cells (ESC) have the capacity to self renew infinitely It is also relatively easy to generate sufficient cells for clinical use However, immune rejection (Heng et al, 2004) of the ESC derived tissue poses a

major hurdle in the use of these cells Although lifelong immunosuppression can be

possible, it would be preferable to design bioengineered products that would be

tolerated by the recipients without the use of immunosuppressive drugs With reports

of reprogramming of adult somatic cells to ESC like cells (Yu et al, 2007) concerns

over immune rejection could be eliminated very soon

Other challenges in the use of ESC include controlling lineage specific

differentiation and elimination of residual stem cells Thus to obtain a purified

population of osteoblasts from ESCs a good understanding of the mechanism

underlying the lineage specific differentiation of hES cells is mandatory

1.5 Human Embryonic stem cells

As the name suggests, the human embryonic stem cells are derived from the

early embryo These cells have the capacity to self renew and can be directed to

differentiate into derivates of all the three germ layers However, in an intact embryo

pluripotent undifferentiated cells are short lived Thus stable maintenance of

undifferentiated ESCs is only possible in vitro cultures

1.5.1 Derivation of HES cells

Human embryonic stem cells are derived from the inner cell mass of the

developing blastocyst- stage embryo The blastocyst is structurally a hollow sphere

composed of the outer trophoblast, which develops into placenta and its supporting

structures, the blastocoel, a fluid filled cavity inside the blastocyst and the inner cell

mass (ICM) which gives rise to all cell types within the embryo

1994 (Bongso et al 1994); however these cells were kept in culture for only a few

passages The first derivation of a hES cell line from ICM of a blastocyst was published in 1998 by Thomson et al

To date hES cells have been derived from a variety of sources, including morula stage embryo, single human blastomeres and later blastocyst stage embryos

1.5.2 Expansion and maintenance of pluripotency of HES cells

Under the microscope, undifferentiated hES cells have a distinct morphology They appear in tightly packed colonies with sharp borders Most hES cells are heterogeneous as they contain both undifferentiated stem cells and some differentiated derivatives Long term stability of hES cells is a requirement that has to

be met for prolonged expansion of these cells in culture Specialized culture conditions and culture medium are required to maintain its undifferentiated phenotype

In vitro, hES cells are cultured on feeder cells which produce various factors

essential for the growth and maintenance of hES cells in their pluripotent state Mouse embryonic fibroblasts (MEF) have been successfully used for this purpose The propagation medium consists of DMEM/F-12 supplemented with Knockout serum replacement, supplemented with basic fibroblast growth factor to sustain the undifferentiated stage of the hES cells

In addition to conventional feeder based cultures, feeder-free culture systems are also widely in use Matrigel is a soluble basement membrane extract of the Engelbreth-Holm-Swarm tumor that gels at room temperature to form a basement membrane (Kleinman et al, 1986) The major components of Matrigel matrix are laminin, collagen IV, entactin and heparan sulfate proteoglycan (Kleinman et al,

1982) Growth factors, collagenases, plasminogen activators and other undefined components have also been reported in Matrigel matrix (Vukicevic et al, 1992) Xu et

al reported a culture system that utilized matrigel and conditioned media of mouse embryonic fibroblasts containing animal component-containing serum replacement and basic fibroblast growth factor, bFGF, for hESC culture

Studies on molecular signaling of hESCs have revealed various pathways that are crucial for the maintenance of pluripotency These include FGF, TGFβ/activin/ nodal, BMP, Wnt pathways (Sato et al, 2003)

Fibroblast growth factor2 signaling is crucial for the self renewal of hES cells The mechanism of FGF2 signaling include induction of supportive factors secretion

in MEF, up-regulation of major genes expressed in TGFβ/activin/nodal signaling and inhibition of BMP signaling (Gerber et al, 2007)

The activation of the TGFβ/activin/nodal signaling through SMAD2/3 is associated with pluripotency and is required for the maintenance of the undifferentiated state in hES cells (James D et al, 2005)

The Wnt pathway is also reported to enhance cell survival and proliferation transiently in hES cells (Dravid et al, 2005; Cai et al, 2007)

With the identification of factors responsible for the maintenance of ESC pluripotency, defined feeder free culture media, TeSR1, has been developed by Ludwig and colleagues in which defined growth factors ( bFGF, TGFβ, aminobutyric acid, pipecolic acid and lithium chloride) have been added to replace the dependence on mouse embryonic fibroblasts-derived media The modified TeSR-1 (mTeSR1) used for propagation of hESCs also includes some animal sourced proteins yet retains the advantage of being serum free

1.5.3 Characterization of hES cells

Several markers are used to identify the pluripotency in hES cells These include surface markers such as glycolipids, glycoproteins, stage-specific embryonic antigens (SSEA) 3/4, tumor rejection antigens (TRA)-1-60, TRA-1-81 (Amit et al, 2000; Reubnoff et al, 2000; Rosler et al 2004) surface antigens: AC133, CA117, CD135, CD9 and transcription factors oct3/4, Sox-2 and Nanog (Hoffman et al, 2005)

About 16 novel genes have been identified that are unique to hES cells These include zinc finger proteins, GSH1 homeodomain protein containing a HOX domain and ESC-specific transcription factor Nanog (Bhattacharya et al, 2004) Telomerase activity, mitochondrial metabolism, genomic stability and epigenetic stability are some of the other standards in characterizing hES cells (Loring et al 2006)

Human embryonic stem cells have shown to retain a stable diploid karyotype and continuously express high levels of telomerase during long term cultures (Zeng and Rao, 2006)

HESCs capability to differentiate into all the three germ layers can be

demonstrated by their ability to form embryoid bodies in vitro (Wobus et al, 2001) and teratoma in vivo (Thomson et al, 1998)

1.6 Differentiation of hES cells into osteogenic lineage

The first report on osteogenic differentiation of ESCs was based on mouse ESCs and was published in 2001 The mouse ESCs were induced to form embryoid bodies following which they were directed to osteogenic differentiation (Buttery et al, 2001)

In vitro differentiation of ESCs into osteoblasts needs the addition of some supplements into the culture media These include β-glycerolphosphate, ascorbic acid and vitamin D3 which are needed for matrix formation and mineralization Stimulation of the culture medium with dexamethasone resulted in increased bone nodule formation (Beilby et al, 2004)

Similar methods have been followed for osteogenic differentiation of human

ES cells (Sottile et al 2003, Bielby et al 2004) Karp et al demonstrated the formation

of bone nodules in 10 to 12 days without the EB step Ahn et al cocultured hES cells with primary bone derived cells and showed that primary bone derived cells are able

to induce differentiation of hES cells into osteoblasts without the addition of exogenous factors

Zur Nieden et al studied the expression pattern of osteoblastic markers during the differentiation of ES cells The pattern of expression of marker genes can be related to phases of osteogenesis Three phases include (1) proliferative phase (2) matrix deposition phase (3) mineralization phase Type1 collagen mRNA is expressed at the end of proliferative phase and during matrix deposition phase Osteopontin mRNA is expressed at the end of matrix deposition phase and at the beginning of the mineralization phase BSP, Cbfa1/Runx2 are expressed during the mineralization phase indicating the presence of mature osteoblasts Osteocalcin mRNA is considered an essential marker of the mineralization state

It was suggested that the length of time that differentiating hES cells are maintained in cultures (about 21 days) is in accordance with the time scale for osteogenic differentiation pathway of human primary osteoblasts and MSC cultures (Buttery et al, 2001) The capacity of the hES cells to differentiate into osteoblasts

and their ability to mineralize can be detected through staining procedures such as alizarin red or von Kossa

1.7 In vivo bone formation using Stem Cells

Bone formation through in vivo implantation of the stem cells that are directed

to osteogenic differentiation are being studied and reported

First clinical reports of human MSC loaded onto a scaffold for trial on patients was by Quarto et al in 2001 The implants showed good osteointegration of newly formed bone However the results were solely based on radiographic examinations and biopsies were not taken Meijer et al (2007) have mentioned about their pilot study in which 10 patients were treated with cultured MSCs loaded on coralline hydroxyapatite (HA) scaffolds, for intra-oral defects Only in one patient were they able to establish that the newly formed bone was due to the implanted cells

There are also reports on in vivo osteogenic potential of hES cells, when

implanted into animal models Bielby et al (2004) showed that culture conditions that

resulted in vitro osteogenic differentiation of hES cells also yielded mineralized tissue when implanted in vivo into the dorsum of SCID mice

Bone formation by endochrondal ossification was achieved in vivo in

immunodeficient mice, by using mouse ES cell derived cartilage constructs as base

material (Jukes et al, 2008) A cartilage matrix was formed in vitro by using mouse

ES cells seeded on to scaffolds When these tissue engineered constructs were implanted subcutaneously, the cartilage matured became hypertrophic, calcified and was ultimately replaced by bone in 21 days When the authors followed the same

approach with hESCs, they found that the chondrogenic potential of hESCs was

insufficient to form cartilage in vitro and bone in vivo

Tremoleda et al in 2008 compared the in vivo osteogenic potential of hES cells with that of hMSC They demonstrated that bone formed by in vivo directed

differentiation of hESCs in diffusion chamber model had no obvious qualitative difference to bone formed by hMSC

A comparison of osteogenesis of hES cells within 2D and 3D culture systems demonstrated that the osteogenic differentiation of hES cells is enhanced in a 3D

culture system compared to a 2D culture environment in vitro When these

cell-scaffold constructs were seeded onto cortical defects created on rabbit bone, they yielded bone after about 4 weeks (Tian et al 2007)

In our study we have compared in vivo formation of bone from osteoblasts

differentiated from hES cells with a human somatic osteoblasts cell line HES cells differentiated osteoblasts are genetically young bone forming cells which can ultimately be used for bone reconstruction This study would help us establish the efficacy of applying hESCs-dervied osteogenic cells in bone formation

CHAPTER 2 MATERIAL & METHODS

2 MATERIAL & METHODS

2.1 Culture and maintenance of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) used in this study were the H1 cell lines (isolated and established at the University of Wisconsin) with normal karyotype These H1cells were cultured on a layer of murine embryonic fibroblasts (MEFs) as described in chapter 1 Culture plates (6 well plates) were first coated with 0.1% (v/v) gelatin (Sigma) and left overnight The next day the plates were seeded with mitomycin-C inactivated MEFs at a density of 1.4 million cells per 6-well plate These MEFs were allowed to attach for a day prior to use as feeder layers Then H1 cells were propagated on this layer of MEF cells

H1 cells were maintained in hESC culture media comprised of Dulbeccos modified eagles medium/ F12 (DMEM/F12, Gibco BRL, Grand Islands, NY, USA) supplemented with 20% (v/v) KnockoutTM Serum Replacement (Gibco BRL), 1% (v/v) nonessential amino-acids (Gibco BRL), 1mM L-glutamine (Gibco BRL), 0.1nM

β-mercaptoethanol (Sigma) and 4 ng/ml basic fibroblast growth factor (bFGF; Gibco, BRL) Cultures were kept in an undifferentiated state at 37 C and in a 5% CO2 atmosphere and checked visually under a light microscope Culture media was changed every day H1 cells usually reached confluence by day five These cells were then removed from the culture dish by incubation with 1 mg/ml collagenase type-IV (Gibco BRL) for about 3 to 5 minutes at 37 C followed by mechanical scraping of the culture plates The H1 cells were then re-plated at a splitting ratio of 1:6 to new 6-well culture plates seeded with MEFs

The MEFs were prepared by expanding them in basic media which consisted

of DMEM (Sigma, St.Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (FBS; Hyclone, USA) MEFs were passaged at 1:5 splitting ratio up to five passages after which they were inactivated with 10 µg/ml mitomycin-C (Kyowa,Tokyo, Japan) for 2 hrs at 37 C before use as feeder cells

2.2 Initiation of osteogenic differentiation in hESCs

To initiate differentiation, H1 cells were obtained after treatment with collagenase Approximately 500,000 cells were seeded onto each well of a 24-well culture plate The 24-well culture plate was coated with gelatin the previous day and left overnight The seeded cells were first left in basic media (DMEM with 10% FBS) for a day and then changed to culture media treated with osteogenic supplements The osteogenic media consisted of basic media supplemented with 50 µg/ml ascorbic acid (sigma), 10 mM β-glycerolphosphate (sigma), 10-8 M vitamin D3 and

10-6M dexamethasone (Sigma) Culture media was changed once every 2 days until day 21 The cells were maintained in incubators at 37 C and in a humid 5% CO2 atmosphere

2.3 Human fetal osteoblasts and their culture conditions

Human fetal osteoblasts (hFOB; CRL- 11372, ATCC, VA, USA) were used as control cells in this study Human fetal osteoblasts were cultured in DMEM/F-12 (Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone,

UT, USA) and 0.3 mg/ml Geneticin (G418 sulfate, Gibco) All cultures were incubated at 34 C in 5% CO2 according to recommendation of ATCC A temperature

of 34 C was considered more optimal for the proliferation of hFOB instead of the

usual 37 C (Ge.Z et al, 2008) Culture media was changed once every three days Osteoblasts were trypsinized when confluent and replated at a splitting ratio of 1:3

For differentiation about 500,000 cells were plated onto each well of a 24-well plate The cells were maintained for a day in hFOB culture media and then changed

to osteogenic culture media The osteogenic culture media consists of DMEM/F-12 supplemented with 10% FBS, 50 µg/ml ascorbic acid, 10mM β- glycerolphosphate

10-8 M vitamin D3 and 10-6 M dexamethasone Cultures were maintained in osteogenic differentiation medium for 21 days with the media changed once every three days

2.4 Methods for assessment of in vitro osteogenic differentiation

2.4.1 RNA extraction and c-DNA synthesis

Total RNA was collected from the osteogenic cultures after 7 and 21 days using RNeasy Mini Kit (Qiagen, Chatsworth, USA) following the manufacturer’s instructions Samples were taken from undifferentiated H1 cells and osteo-differentiated H1 & hFOB cultures Samples were treated with RNase-free DNase ŀ (Qiagen) to remove any genomic DNA contamination The extracted RNA was quantified using Nanodrop (Nanodrop Technologies, Wilmington, DE) About 500 ng

of mRNA was used to synthesize the required cDNA cDNA synthesis kit (Bio-Rad, Hercules, CA) was used for this purpose

2.4.2 Conventional Polymerase Chain Reaction

Conventional PCR was performed using PCR thermal cycler, Mycycler Rad) Samples were amplified for 35 cycles at 95 C for 5 minutes, 35 cycles at 95 C for 30 seconds, 55-65 C for 45 seconds, 72 C for 1 minute, followed by 72 C for 5

(Bio-minutes β-actin was the house keeping gene used to normalize the PCR reactions Electrophoresis of the amplified products was run on 2% agarose gel The products were stained with ethidium bromide and viewed using Light Imaging System (Bio-Rad) PCR primers, annealing temperature, primer sequence and their product size are listed in Table 1

2.4.3 Detection of calcium deposition by alizarin red staining

As an indicator of mineralization within both the H1 and hFOB osteogenic differentiation cultures, calcium deposition was analyzed by alizarin red staining at 7,

14 and 21 day time points 2 g of alizarin red S (sigma) was dissolved in 100 ml of de-ionized water The pH of the solution was adjusted to 4.2 using 0.5% ammonium hydroxide The solution was filtered before use

After washing with PBS the osteogenic cell cultures were fixed in 4% paraformaldehyde for 30 minutes at room temperature Then the cultures were again washed with PBS and stained with alizarin red for about 5 minutes The dye was removed and the cell cultures washed with deionized water The mineralization was observed under the microscope The calcium salts within the bone nodules stained red while the collagenous extra cellular matrix turned yellow

2.4.4 Hoechst DNA quantification assay

Samples were taken on day 7, 14 and 21 of osteogenesis in both hESC and hFOB osteogenic differentiation DNA concentration was assessed to compare the cell density during the mentioned time points

10 mg/ml calf thymus DNA was diluted in 1xTNE buffer composed of 10 mM Tris Base, 10 mM EDTA and 100 nM NaCl with pH adjusted to 7 Twelve different

concentrations of calf thymus DNA was used to establish standard curve for DNA content 10 µl of each sample was diluted with PBS to a final volume of 100 µl followed by addition of 100 µl of Hoechst33258 Dye for fluorescence measurement

by fluorescence plate reader (Tecan Safire, Austria) at 355 nm excitation and 460

nm emission DNA concentration in each sample was extrapolated from the DNA standard curve

2.5 Embedment of H1 & hFOB cell in Extracel TM

About 500,000 cells of both H1 and hFOB were grown in Extracel TM(Glycosan Biosystems, Salt Lake City, Utah) Extracel is made up of three biocompatible components: Gelin-S, Glycosil and Extralink Hydrogel was formed when the crosslinking agent, Extralink™ was added to a mixture of Glycosil™ and Gelin-S™ which also contained the cells This mixture was then placed in culture plates and allowed to gel at 37 C Gelation occurred in about twenty minutes. Then the respective culture media were added to each of the wells holding the cell-gel construct Media was changed every day for the H1 cultures and once in two days for hFOB cultures The cultures were maintained in gel for a week and then assessed for cell viability Extracel is intended to be used as a cell carrier for seeding

cells into the scaffolds for in vivo experiments

2.5.1 Assessment of cell viability in Extracel TM

2.5.1.1 FDA/PI staining

Cell viability in Extracel was assessed by Fluorescein diacetate/ Propidium iodide (FDA/PI) staining 12 µM stock solution was prepared by dissolving of 5 mg FDA (Sigma) in 1 ml of acetone Then FDA working solution was prepared by adding

0.04 ml of stock to 10 ml of dulbeccos phosphate buffered saline (DPBS) 1.5 µM working solution was prepared by dissolving 1 mg of PI (Sigma) in 50 ml of DPBS for use

After a week in Extracel culture, the culture media was removed and the cells (both H1 and hFOB) were stained with 0.1 ml of FDA (2 µg) and 0.03 ml (0.6 µg) of

PI for about 3 mins and then placed in ice

2.5.1.2 Confocal microscopy

Confocal microscopic analysis of the FDA/PI stained cells embedded in Extracel was carried out using a Ziess Fluoview laser confocal microscope The wavelength of the excitation and emission for FDA were 488 nm and 505-503 nm and for PI were 543 nm and 585 nm Image processing was completed using software supplied by the confocal microscope’s manufacturer

2.6 In vivo experiments

2.6.1 Culture of H1 cells in Matrigel

For in vivo experiments H1 cells cultured in feeder-free culture system were

used Confluent H1 cells grown on feeder layer were removed from the culture plastics after incubation with collagenase The H1 cells were replated into mTeSR1TM (Stemcell Technologies Inc) The new culture plates were coated with BD MatrigelTM (BD Biosciences) an hour prior to replating of H1 cells Media was changed once every day

Cells were removed from the culture plastics when reaching confluence after incubation with dispase (Stemcell Technologies) for 7 minutes at 37 C and mechanical scraping The cells were then replated onto new Matrigel-coated culture

plates The H1 cells were cultured in feeder-free culture for 2 weeks prior to implantation in vivo

2.6.2 Pre-differentiation of cells in scaffolds prior to in vivo implantation

About 500,000 cells of either H1 or hFOB were seeded into each of the PLGA scaffold (Bio-scaffold International, Singapore) using ExtracelTM as the cell carrier The cell-scaffold constructs were left in their respective culture media for a day After

24 hrs, osteogenic differentiation was initiated in these cells by replacing the culture media with their respective osteo-differentiation media The cell scaffold constructs

were maintained in osteogenic culture media for two weeks before implantation in vivo Media was changed once every two days

2.6.3 Labeling of differentiating cell with CFDA-SE

Vybrant R CFDA SE kit was used for in vivo tracing of the cells in this study

10 mM stock solution was prepared by dissolving 500 µg Carboxy-flurorescein diacetate, succinimidyl ester (CFDA SE) powder into 90 µl of dimethylsulfoxide (DMSO) This stock solution was then diluted in PBS to the required working concentration of about 15 µM

The cell-scaffold constructs were maintained in osteogenic differentiation media for about 2 weeks before labeling Culture media was removed from the dishes The cells were incubated for 15 minutes in prewarmed (37 C) PBS containing CFDA SE This loading solution was then replaced with fresh prewarmed (37 C) osteogenic differentiation media The cell scaffold constructs were incubated for about 30 minutes in 37 C before observation under the fluorescent microscope

Then the cell scaffold constructs were left in the incubator for a day before implantation in vivo

2.6.4 In vivo grouping

A total of 6 SCID mice were used for in vivo experiments, with each mouse carrying 5 - 6 samples of scaffold-cell constructs Three of the mice carried test samples which were scaffold-hESC constructs, while the other three carried positive control samples which were scaffold-hFOB constructs One mouse each from the test and control groups were sacrificed at 2nd, 4th and 7th weeks

Group Sample 2 nd week

sacrifice

4 th week sacrifice

7 th week sacrifice

Total

Test PLGA scaffold

+ cell carrier +d-hESC

Seven weeks old male and female SCID mice were used in this study The animals were acclimatized to the animal holding unit one week prior to the surgery The SCID mice were anasthetized with an intraperitoneal administration of AHU CRU mixture (Ketamine + medetomidine) A one centimeter incision was made on the dorsum of the mice Then cell-scaffold constructs (H1 & hFOB osteodifferentiated cultures) were transplanted subcutaneously into both the sides on the dorsal surface The grouping was done as mentioned in the above table The