Liver regeneration and tissue engineering

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA

LIVER REGENERATION AND TISSUE

ENGINEERING

P

EDRO

M

IGUEL

A

LMEIDA DE

M

ATOS

B

APTISTA

D

F

(B

F

)

II

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA

LIVER REGENERATION AND TISSUE

ENGINEERING

P

EDRO

M

IGUEL

A

LMEIDA DE

M

ATOS

B

APTISTA

Dissertation Supervisors:

Prof. Doutor Anthony Atala

Prof. Doutora Maria Henriques Ribeiro

D

F

(B

F

)

III

ACKNOWLEDGEMENTS

-To the 3rd Gulbenkian PhD Program in Biomedicine and everyone that somehow belongs and contributes to it. If it wasn’t for them, I wouldn’t have even started this amazing journey.

-To the Wake Forest Institute for Regenerative Medicine (WFIRM), my Cradle to Science. I’m in debt to all the people that I met there. Their insight, their constructive criticism… our broad scientific discussions over lunch or coffee, your direct contribution to my work… you won’t be forgotten.

-To “Fundacao para a Ciencia e Tecnologia”, Portugal. For all the financial support that they provided and made this dissertation possible – Doctoral Scholarship - SFRH/BD/11802/2003.

-To Dr. Anthony Atala, our “big boss”. Without his leadership, vision and tireless effort building this lab we simply wouldn’t be here. Our Science simply wouldn’t be…

-To Dr. Shay Soker, my mentor and “boss”. Your friendship, counseling, help, guidance and mentorship were invaluable. No surprise that what I achieved, we achieved together. No surprise that what we achieved is now a PhD thesis. I really owe you a lot…

-To Prof. Doutora Maria Henriques Ribeiro, my national supervisor. Thank you for your constant patience, precious help throughout these years and your priceless advice in everything that happened here and before... Without you, I wouldn’t be having the joy of this moment.

-To the faculty of the WFIRM (Dr. Mark Furth, Dr. Mark Van Dyke, Dr. James Yoo, Dr. Koudy Williams, etc) that always helped me immensely with their creative thinking, constructive

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criticism and vast knowledge. I improved so much with your example and advice. Thank you so much for caring.

To my dear, dear friends. Without you, life here wouldn’t have been so fun and interesting. Home sickness hit me sometimes… but you were always there for whatever it took (Dawn, Daniel, Tamer, Akira, DJ Lee, Luiz, Paulina, Fernanda, Julie, Anna, Lauren, Ken, Yaz, Sergio, Simone, etc, etc). For you, for all of you… Muito obrigado.

-To all those people… that somehow and at some point, contributed to the work presented in this dissertation. You have my deepest gratitude.

-Finally, to My Family. To my brother and sister-in-law for their words and support; to my nephew, my hope for a better future; to my grandparents, for their unconditional love and encouragement, showing me at every moment the true meaning of soul and body “regeneration” from the height of their 90 years old; and foremost, to my parents. Without you, I wouldn’t have endured, I wouldn’t have come, I wouldn’t have prevailed. My absence has generated all that sorrow and “saudades”… but with the promise of coming back home soon, I hope you understand that all these I accomplished… was ultimately for you.

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VI INDEX Abbreviations XI Abstract XII Summary XIII Resumo XV Chapter 1 – Introduction

1.1 Liver Disease and Liver Transplantation 2

1.2 Liver Anatomy and Physiology 4

1.3 Liver Functional Unit – the Acinus 6

1.4 Hepatocytes 8

1.5 Endothelial Cells 9

1.6 Liver Regeneration 10

1.7 Bioartificial Liver Devices 12

1.8 Liver Tissue Engineering 13

1.9 Objectives 14

1.10 Bibliography 16

Chapter 2 – Generation of a 3D Vascularized Liver Bioscaffold for Whole Organ Engineering

2.1 Introduction 22

2.2 Materials and Methods 24

2.2.1 Liver preparation 24

2.2.2 Decellularization 24

2.2.3 Matrix characterization 25

2.2.4 Biodegradation assay 25

VII 2.2.6 Bioscaffold endothelial cell seeding and bead perfusion 26

2.2.7 Bioscaffold seeding with HepG2 and endothelial cells 26

2.2.8 Implants 26

2.2.9 Transplantation 27

2.3 Results 28

2.4 Discussion 40

2.5 Bibliography 44

Chapter 3 – Perfusion Cell Seeding Optimization of the Decellularized Liver Bioscaffold

3.1 Introduction 50

3.2 Materials and Methods 51

3.2.1 Liver Bioscaffold Preparation 51

3.2.2 Bioreactor Assembly 51

3.2.3 Cell Seeding Optimization 53

3.2.4 Fixation and Tissue Processing 54

3.2.5 Histomorphometric Analysis 54

3.2.6 Histochemical and Immunohistochemical Analysis 54

3.2.7 Rat Primary Hepatocyte Seeding 55

3.3 Results 56

3.4 Discussion 63

3.5 Bibliography 66

Chapter 4 – Human Liver Stem/Progenitor Cells for Liver Tissue Engineering

4.1 Introduction 71

4.2 Materials and Methods 74

VIII 4.2.2 Human Fetal Livers 74

4.2.3 Immunohistochemistry 75

4.2.4 Molecular analysis 76

4.3 Results 78

4.4 Discussion 84

4.5 Bibliography 86

Chapter 5 – hAFS as a Source of Hepatic and Endothelial Progenitors for In Vivo Cell Therapy

5.1 Introduction 90

5.1.1 Development and Characterization of Amniotic Fluid 90

5.1.2 AFS Isolation, Characterization and Differentiation 94

5.1.3 Implantation AFS cells in vivo and in animal models of regeneration 99

5.2 Materials and Methods 100

5.2.1 Teratoma analysis 100

5.2.2 Myoblast Implantation 100

5.2.3 Endothelial Cell Implantation 101

5.2.4 Hepatocyte Implantation 101

5.3 Results 103

5.4 Discussion 107

5.5 Conclusion 108

5.6 Bibliography 109

Chapter 6 – Conclusions and Future Perspectives

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INDEX OF FIGURES AND TABLES

Chapter 1

Figure 1.1 Number of patients in Waiting List vs Transplanted until 2007 in the USA 2

Table 1.1 Liver transplantation waiting list patient characteristics in the USA 3

Figure 1.2 Liver segments anatomy 6

Figure 1.3 Acinar microstructure 7

Chapter 2

Figure 2.1 Preparation of the acellular vascularized bioscaffold 28

Figure 2.2 Histochemical analysis of decellularized liver bioscaffold 30

Figure 2.3 Collagen types I, II, III and IV immunohistochemical staining 30

Figure 2.4 Biodegradation of the AVB in the presence of collagenase II 31

Figure 2.5 Ultrastructure of the liver bioscaffold 32

Figure 2.6 Characterization of patency of the AVB vascular tree 34

Figure 2.7 Re-endothelialization of the liver bioscaffold 35

Figure 2.8 Re-cellularization of the liver bioscaffold 36

Figure 2.9 Rat hepatocytes and mouse endothelial cells implanted in bioscaffold 37

Figure 2.10 Transplantation of liver bioscaffold 38

Chapter 3

Table 3.1 Perfusion cell seeding bioreactor conditions 52

Figure 3.1 Bioreactor Layout 52

Figure 3.2 Macroscopic appearance of the bioscaffold 57

Figure 3.3 Bioscaffold cell density using different flow rates for cell seeding 57

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Figure 3.5 Protein expression analysis of seeded bioscaffolds 59

Figure 3.6 Tissue formation and organization of cells engrafted in the bioscaffold 60

Figure 3.7 Tissue formation of rat primary hepatocytes in the bioscaffold 61

Chapter 4

Figure 4.1 Immunofluorescence of hFL cells seeded in bioscaffold (5 days) 78

Figure 4.2 Determination of hFL cell proliferation by DNA quantification 80

Figure 4.3 Immunofluorescence of hFL cells seeded in bioscaffold (21 days) 80

Figure 4.4 Protein expression of hFL cells seeded in bioscaffold disks 82

Chapter 5

Table 5.1 Flow cytometric analysis of amniotic fluid cells (no expansion) 93

Figure 5.1 Histological analysis of mice teratomas (mES) and testis (hAFS) 103

Figure 5.2 Histological analysis of hAFS cell implantation 104

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ABBREVIATIONS

3D – Three-Dimensional AFS - Amniotic Fluid Stem

anti-GFP – Anti-Green Fluorescent Protein AVB – Acelular Vascularized Bioscaffold BHA – Butylated Hydroxyanisole

DAB - 3,3’ Diaminobenzidine

DMEM – Dulbecco’s Modified Eagle’s Médium DMSO - Dimethyl Sulfoxide

ECM – Extra-cellular Matrix EGM - Endothelial Cell Medium FBS - Fetal Bovine Serum

hAFS – Human Amniotic Fluid Stem Cells HDL - High Density Lipoprotein

HepG2 - Human Hepatocellular Liver Carcinoma Cell Line hFL – Human Fetal Liver

HpSC – Hepatic Stem Cells IL-1 – Interleukin-1

IL-2 - Interleukin-2

MDR – Multidrug Resistant MS1 - Mouse Endothelial Cells NGF - Nerve Growth Factor NO – Nitric Oxide

PBS – Phosphate Buffered Saline PI – Propidium Iodide

RER - Rough Endoplasmic Reticulum SEC – Sinusoidal Endothelial Cells

VEGF - Vascular Endothelial Growth Factor VLDL – Very Low-Density Lipoprotein WHO – World Health Organization

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ABSTRACT

Over 21 million people in the world are estimated to live with chronic liver disease, and about 800,000 expire annually, accordingly with WHO. Liver transplantation remains today the definitive treatment for end-stage liver failure. However, the gap between organ donation and the number of patients in the waiting list for a liver keeps widening. The shortage of organs has stimulated the research for alternatives in end-stage liver disease. Cell therapies with some degree of success are today a reality. Nevertheless, the goal of generating a whole liver in vitro remains elusive. The purpose of the work of this dissertation was to develop a new approach to liver tissue engineering that would allow the generation of significant mass tissue readily implantable and/or transplantable. Cell sources for tissue engineering applications are of vital importance. Our research focus on the use of hFL progenitor cells in combination with the liver bioscaffold and on a new promising source of fetal stem cells from amniotic fluid, in liver therapies and tissue engineering.

In our work, we were able to create a novel acellular liver derived bioscaffold with preserved vascular network. This bioscaffold could be efficiently re-cellularized and considerable mass tissue, displaying some hepatic functions, was generated. It was also successfully transplanted to living hosts and perfused with blood. Another goal attained in this work was that the combination of hFL progenitor cells with the liver bioscaffold produced cell engraftment, expansion and differentiation of the hFL cells. hAFS cells also showed engraftment and integration in injured livers, representing a new alternative for in vivo cell therapies and liver regeneration.

In conclusion, this doctoral dissertation clearly demonstrates the successful generation of 3D liver tissue with a novel acellular liver bioscaffold using different cell sources. This potentially represents a new hope for patients suffering of end-stage liver disease.

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SUMMARY

The severity of end-stage liver disease is directly related with the vital role that the liver plays in systemic metabolic homeostasis. Organ transplantation remains today the definitive treatment for end-stage liver disease. Due to the shortness in organ availability, new alternatives have been sought in the last decade. With the dawn of regenerative medicine, cell transplantation using adult hepatic cells has emerged as a potential therapeutic option to treat various severe liver conditions. Although successful, therapies with adult hepatic cells usually fade within several months. The advancement of stem cell biology as also brought new opportunities in cell therapies with newly identified or differentiated stem/progenitor cells. It also increased the opportunities in finally generating a whole liver in vitro able to be readily implanted or transplanted into a host.

In this work, the generation of a novel liver derived biomaterial that could preserve its native vascular network was investigated. We attempted to improve the decellularization of thick tissues and solid organs employing some of the tissue decellularization techniques used for the generation of naturally derived scaffolds. We were succeeded in our attempts on perfusing decellularization solution through the liver vascular system instead of simply shaking the organ with the decellularization solutions relying only on reagent diffusion. The outcome was the generation of a novel acellular liver derived bioscaffold which preserves its native vascular network. This allows the perfusion of culture media and cell seeding through the decellularized organ vasculature reaching virtually every position in the thick 3D bioscaffold. Oxygen and nutrient diffusion limitations are now overcome by the use of the decellularized organ native vascular system, allowing in vitro generation of dense 3D tissue.

The optimization of the re-cellularization process was carried out by seeding the cells with culture media perfusion. The use of different perfusion flow rates allowed us to find the optimal

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conditions to seed these liver bioscaffolds with different types of cells, which generate high cellular density 3D tissues expressing characteristic hepatic functions.

In order to further enhance hepatic tissue generation, we investigated the use of hFL progenitor cells in combination with the liver bioscaffold. The use of these liver progenitor cells are a valuable resource for liver tissue engineering and cell therapies. Preliminary experiments indicated that hFL progenitor cells seeded in bioscaffold disks engrafted, proliferated and differentiated in hepatocytic and biliary cell lineages.

Finally, we investigated the use of hAFS cells in several in vivo regeneration models. Our work confirmed the non-tumorigenic potential of these cells and demonstrated their integration and differentiation in injured muscle and response to angiogenic stimuli. Moreover, these cells were able to engraft in damaged livers. The in vivo multipotenty exhibited by hAFS cells is encouraging and confirms their potential in regenerative medicine applications.

Overall, this dissertation work emphasizes the relevance of a new organ decellularization method able to generate liver acellular bioscaffolds with preservation of a functional vascular network. This allows culture media perfusion with 3D tissue generation beyond the oxygen and nutrient diffusion limits. Furthermore, we present evidence that hFL progenitors in combination with the liver bioscaffold and hAFS cells offer new possibilities in liver tissue engineering and cell therapies of end-stage liver disease.

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RESUMO

A gravidade das doenças hepáticas terminais está directamente relacionada com o papel vital que o fígado desempenha na homeostase metabólica sistémica. A transplantação de orgãos permanece ainda hoje como o único tratamento definitivo para a doença hepática terminal. Devido à escassez de orgãos, novas alternativas têm sido investigadas na última década. Com o despontar da medicina regenerativa, a transplantação de células hepáticas adultas tem emergido como um opção terapêutica válida no tratamento de várias patologias hepáticas graves. No entanto, embora com algum sucesso, o efeito terapêutico diminui e desaparece ao fim de alguns meses. Os recentes avanços no conhecimento da biologia das células estaminais, nomeadamente na identificação e caracterização, trouxeram novas oportunidades de terapêuticas celulares com recurso a populações de células estaminais/progenitoras. A possibilidade de finalmente gerar um fígado totalmente in vitro passível de ser facilmente implantado ou transplantado, também aumentou.

Neste trabalho, foi investigada a geração de um novo biomaterial derivado do fígado preservando intacta a sua rede vascular. Para tal, foram empregues algumas das técnicas de descelularização de tecidos, usadas na obtenção de matrizes naturais, e efectuada a optimização destes processos na descelularização de tecidos mais espessos e orgãos sólidos. O processo revelou-se eficaz quando soluções de descelularização foram perfundidas pelo sistema vascular do fígado em alternativa à simples agitação do órgão imerso nas soluções de descelularização e cujo princípio se baseia na difusão dos reagentes pelo órgão. O resultado foi a obtenção de uma nova matriz acelular derivada do fígado e que preserva o seu sistema vascular nativo. Esta matriz permite a perfusão de meio de cultura e de células através da rede vascular do orgão descelularizado, permitindo virtualmente atingir qualquer posição da espessa matriz 3D. As limitações de difusão de oxigénio e de nutrientes são assim ultrapassadas pela utilização da rede vascular nativa do orgão descelularizado com a geração de denso tecido 3D in vitro.

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Foi também efectuada a optimização do processo de re-celularização, através da perfusão das células com meio de cultura pela rede vascular do orgão descelularizado. A utilização de diferentes velocidades de perfusão da suspensão celular permitiu determinar as condições ideais para a re-celularização destas matrizes acelulares com diferentes tipos de células. Isto permitiu também gerar tecido 3D densamente re-celularizado e com expressão de marcadores hepáticos característicos.

Para melhorar a geração de tecido hepático, foi investigado o uso de células progenitoras humanas obtidas a partir de fígados fetais em combinação com a matriz acelular. O uso destas células progenitoras constitui sem qualquer dúvida um recurso determinante na engenheria de tecidos do fígado e em terapias celulares. Ensaios preliminares revelaram que estas células progenitoras humanas cultivadas em discos de matriz acelular se estabeleceram, proliferaram e diferenciaram em células de linhagem hepática e biliar.

Finalmente, foi investigado o uso de células estaminais humanas obtidas a partir do fluído amniótico em vários modelos de regeneração in vivo. As experiências confirmaram a natureza não tumorigénica destas células e demonstraram a sua integração e diferenciação em modelos de lesão muscular e de angiogénese. Adicionalmente, estas células demonstraram capacidade para se fixar em fígados danificados. A plasticidade exibida in vivo por estas células é notável e confirma o seu potencial em medicina regenerativa.

Globalmente, nesta dissertação de doutoramento destaca-se a relevância de um novo método de descelularização de orgãos passível de gerar matrizes acelulares derivadas a partir do fígado com preservação de uma rede vascular funcional. A perfusão de meio de cultura e a geração de tecido 3D permite ultrapassar os problemas de difusão de oxigénio e de nutrientes. Por último, a

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utilização de células progenitoras humanas, obtidas a partir de fígados fetais em combinação com a matriz acelular, e de células estaminais humanas, derivadas a partir do fluído amniótico, apresentam potencialmente novas oportunidades na engenharia de tecidos do fígado e nas terapias celulares na doença hepática terminal.