Regenerative Medicine of Pancreas

Nature Biotechnology,23:857

楊卿堯 醫師

Cell Therapy Center, Department of Surgery, National Taiwan University Hospital

2011-08-10 暑期再生醫學學程

Topic

1. Pancreas Anatomy2. Embryology and development3. Histology and Islet composition 4. β-cell function, insulin secretion, and glucose

regulation5. Diabetes and treatment6. Regeneration of islet-like cells7. Human islet transplantation

Anatomy

2006 McGraw-Hill Higher Education online learning center

Embryology

The Deveolping Human Moore KL 4th ed 1988 Saunders

(5wks-8wks)

The role of islet transcription factors in endocrine differentiation in the

developing pancreas

Histology

• In health, the human pancreas contains approximately one million islets, each containing approximately two thousand beta-cells.

• The beta-cells constitute 1.5% of the total pancreatic mass (1–2 g in total).

• While in humans, an up to 40% loss of beta-cells can be tolerated without a significant deterioration of glucose tolerance, a further reduction in beta-cell mass leads to hyperglycemia.

Islets

•

Normal islets

Islets in type I DM patient

Islets composition

•

Islets show striking interspecies differences

•

77%

55%

18% 38%

Promoter region of insulin gene

• Insulin gene: 11p15.5• PDX-1: homeodomain transcription factor

pancreatic/duodenal homeobox-1

-340bp

Insulin synthesis

Preproinsulin

21 a.a.

30 a.a.

Glucose control of insulin secretion

Diabetes mellitus

• Classification: Type I Autoimmune diseaseType II insulin resistence

• Hyperglycemia:Pre-diabetes: fasting plasma glucose: 100-125 mg/dlDiabetes: fasting glucose level > 126 mg/dl

• Complications of diabetes:

Regenerative Medicine

• Definition:

Regenerative medicine is an emergingfield that approaches the repair orreplacement of tissues and organs byincorporating the use of cells, genes orother biological building blocks along withbioengineered materials and technologies.

Stem cells and progenitor cells

Feature Stem cell Progenitor Cell

Self-renewal Unlimited Can be limited

Plasticity Pluripotent Unipotent or Multipotent

http://en.wikipedia.org/wiki/Pluripotency�http://en.wikipedia.org/wiki/Unipotent_cell�http://en.wikipedia.org/wiki/Multipotency�

Sources of pancreatic β-cells

Strategies to produce β -cells suitable for transplantation

Abcd

• 123

Strategies for generating supplies of β -cells

1. Expansion of primary β -cells2. Pancreatic progenitor cells3. Transdifferentiation of non-pancreatic cells4. Embryonic stem cells

β -cell turnover in adult humans

New β -cell formation apoptosis

Regeneration of β -cells in the pancreas

1. Expansion of primary β -cells2. Pancreatic progenitor cells

Rodent model

•

Transdifferentiation

• Definition: The ability of multipotential adult stem cells to cross lineage boundaries and to give rise to cells not normally found in the organs or tissues of residence.

• For example, bone marrow stromal cells can generate skeletal muscle, and neural stem cells can give rise to blood cells, cardiac muscle and oval hepatocytes.

Possible mechanisms for pancreatic transdifferentiation

•

Embryonic stem cells

• Human ES cells --- > insulin secreting cells (in vitro)• Mouse ES cells ---- > islet like clusters (in vitro)

Strategies to obtain β cells from organ-specific stem or progenitor cells

Generation of insulin-expressing cells from pluripotent cells

臨床胰島細胞移植之現況與新進展

Advances and Current Status of Clinical Islet Transplantation

楊卿堯

台大醫院 外科部 細胞治療中心

2011-08-10

Diabetes Research InstituteSchool of Medicine, University of Miami

Diabetes Research Institute

Dr. Camillo Ricordi （5億 5千萬新台幣/ 年）

1. What is islet isolation and transplantation?2. History of islet transplantation3. Human islet isolation and transplantation

procedure4. Current outcome and advances of islet

transplantation

Topics

What is Islet Isolation and Transplantation?

History of Islet Transplantation

• 1893 First attempted islet xenograft using sheep pancreas fragments by English Surgeon Watson Williams

• 1922 Discovery of insulin by Banting and Best• 1966 First pancreas Tx by Kelly and Lillehei, U of Minnesota

• 1972 First rodent model of islet isolation and Tx by Lacy• 1977 First alloTx using pancreatic tissue fragments

(islets)• 1986 Ricordi chamber, automated method• 1993 COBE continuous gradient purification method• 1997 Digestion using Liberase• 2000 Edmonton Protocol by Shapiro, U of Alberta

1. Use steroid-free immunosuppressants: sirolimus, low dose tacrolimus, daclizumab

2. Infusion adequate islet mass (> 10,000 IEQ/Kg) from multiple donors

Islet Isolation Procedure

1. Clean pancreas2. Cold perfusion with enzyme3. Warm digestion4. Dilution5. Pre-purified islets collection6. Purification with COBE7. Preparation for Tx

1. Pancreas cleaning & cannulation

2. Pancreas cold perfusion

3. Pancreas digestion

Automated islet isolation method

Islet release after warm digestion

6 min

8 min

10 min

4. Dilution

5. Islet collection and preparation for purification

Pre-purification product

6. Purification with continuous gradients method

COBE 2991

Post-purification islets

# Islet enumeration & purity assay

Yield (IEQ) Purity(%)

# Viability assay

Islets on transplant day

Tissue Volume

> 1.5Glucose stimulation test < 10 ml

7. Preparation of cultured islet cells for Tx

***Transplant of human islet product into portal vein***

Current outcome of islet transplantation

Allotransplantation Islet aloneIslet after kidney transplantationIslet simultaneous with kidney

transplantation

AutotransplantationChronic pancreatitisPancreatectomy due to benign

pancreas lesion

Recipient inclusion criteria of Islet alone allotransplantation

• 18-65 years• Type 1 DM >5 years• Complicated at least one of following situations,

despite intensive insulin management1. Reduced awareness of hypoglycemia2. Metabolic lability/instability

(>2 episodes of severe hypoglycemia)3. Progressive secondary complications of DM

Donor inclusion criteria

1. Multi-organ donor2. Adequate in situ hypothermia perfusion3. UW for perfusion and cold storage4. < 12 hr cold ischemia time5. Age 25~70 years6. Death caused by head trauma, ICH, brain

infarction, acute occlusive hydrocephalus, primary brain tumor

• 1999-2004• 65 patients with type 1 DM, mean 42.9 years• Mean islet transplantation/procedure: 5783

IEQ/Kg• Mean packed cell volume: 4.4 ml• Mean hospital stay: 2 days• Acute complications: (128 procedure)

15/65 (23%) procedure-related bleeding, 2 laparotomy5/65 (7.7%) thrombus in branches of portal veinTransient elevation of portal pressure and GOT, GPT

Graft survival by C-peptide positivity

Insulin Independence

82%

70%

40%

20%

7.5%

Edmonton Experience (65 subject, still Nov.2004)

94%

HbA1c

6.4%6.7%

9%

Autotransplantation

台大醫院胰島細胞移植計畫推展情況

2005.12-2006.12.

1. Setup islet isolation instruments, materials, equipments for small animal, large animal, and human pancreas

2. Small animal islet isolation and product assessment in vitro, in vivo model

3. NTUH cGTP Lab remodeling

2007.1-2007.12.

1. Large animal and preclinical human pancreas islet isolation and quality control assessment experiments

2. NTUH New cGTP/GMP Cell Therapy Center plan

2008.1- 2008.5

1. CTC center establishment project was postponed

2008.5 – present Islet Biology and Tx Research

Preconditioning research

Islet XenoTx project

Human pancreas donor characteristics

Table 1.

*HTK solution: Histidine-Tryptophan-Ketoglutarate preservation solution

Human islet isolation processing factorsTable 2.

Human islet isolation outcomeTable 3.

* Islet with diameter 150 um equal to 1 IEQ (Islet equivalent number)

Temperature control and monitoring during warm digestion and dilution phase

10 min sampling 12 min sampling

HP 01 HP 02 HP 03

Collection of purified islets

Purified islets and viability assessment

In vivo assessment

Canine islet isolation

Pig donor profilesTable 7.

Islet isolation outcome of pig pancreasTable 9.

Experience of preclinical work in the past years

1. We have successfully isolated qualified human islets with standard operation procedure.

2. We also trained our isolation team members with three canine and three pig islet isolation experiments.

3. Through these preclinical human and large animal islet isolation trial, we validated our islet isolation program.

NTUH New Cell Therapy Center

University of Miami –Diabetes Research Institute 2005, 2006

University of Minnesota- Molecular and Cellular Therapeutics Center 2007

City of Hope, LA- Center for Biomedicine and Genetics 2008

DOH95 project (2006) DOH96 project (2007) DOH96 project (2007)

(2009)

NHRI project (2008-2010)

Small animal model of islet isolation and transplantation translational study

Large animal model translational study

Preclinical human islet isolation trial

Clinical trial of human islet transplantation

Islet transplantation; translational research

Thanks

臺大醫院- 外科部 李伯皇 主任, 副院長 何弘能 教授慶祥細胞治療中心 姚明醫師 蔚萱 怡珍 佳芳

醫研部

動物中心

臺大醫學院- 毒理所 劉興華 所長, 俊發 雅雯 雨芝 景任 姿安醫工所 林峰輝 所長

臺大動物醫院- 郭宗甫 教授

University of Miami School of Medicine-Diabetes Research Institute Camillo Ricordi and Rodolfo AlejandrocGMP Lab Aisha Khan and islet isolation team membersTranslational Lab Antonello Pileggi and members

Hirohito Ichii and Xiaojing Wang

http://cyang88.blog.ntu.edu.tw/

Thanks臺大醫院- 外科部 李伯皇教授, 副院長 何弘能 教授

一般外科 林明燦主任 田郁文教授 蔡孟昆副教授研究助理 穆姵璇 王韻筑 廖珮含 呂杰修細胞治療中心 姚明醫師 蔚萱 怡珍 佳芳 凱暹 昇憲..醫研部動物中心

臺大醫學院- 毒理所 劉興華 所長, 俊發 雅雯 雨芝 景任 姿安醫工所 林峰輝教授

臺大動物醫院- 郭宗甫 教授

NTUH grant, NTUH-ITRI grant , 衛生署轉譯醫學研究計畫

University of Miami School of Medicine-Diabetes Research Institute Camillo Ricordi and Rodolfo AlejandrocGMP Lab Aisha Khan and islet isolation team membersTranslational Lab Antonello Pileggi and members

Hirohito Ichii and Xiaojing WangUniversity of Minnesota City of Hope Hospital

Regenerative Medicine of PancreasTopic AnatomyEmbryologyThe role of islet transcription factors in endocrine differentiation in the developing pancreas�投影片編號 6HistologyIsletsIslets compositionIslets show striking interspecies differencesPromoter region of insulin geneInsulin synthesisGlucose control of insulin secretionDiabetes mellitusRegenerative MedicineStem cells and progenitor cellsSources of pancreatic β-cellsAbcdStrategies for generating supplies of β -cellsβ -cell turnover in adult humansRegeneration of β -cells in the pancreas投影片編號 22TransdifferentiationPossible mechanisms for pancreatic transdifferentiationEmbryonic stem cellsStrategies to obtain β cells from organ-specific stem or progenitor cells�Generation of insulin-expressing cells from pluripotent cells�投影片編號 28臨床胰島細胞移植之現況與新進展�� Advances and Current Status of �Clinical Islet TransplantationDiabetes Research Institute� School of Medicine, University of MiamiDiabetes Research Institute投影片編號 32What is Islet Isolation and Transplantation?History of Islet Transplantation投影片編號 35投影片編號 36Islet Isolation Procedure1. Pancreas cleaning & cannulation 2. Pancreas cold perfusion 3. Pancreas digestion Automated islet isolation methodIslet release after warm digestion4. Dilution5. Islet collection and preparation for purificationPre-purification product6. Purification with continuous gradients methodPost-purification islets# Islet enumeration & purity assay # Viability assay Islets on transplant day 投影片編號 517. Preparation of cultured islet cells for Tx ***Transplant of human islet product into portal vein***投影片編號 54�Current outcome of islet transplantation Recipient inclusion criteria of �Islet alone allotransplantationDonor inclusion criteria投影片編號 58投影片編號 59HbA1cAutotransplantation台大醫院 胰島細胞移植計畫推展情況�Human pancreas donor characteristicsHuman islet isolation processing factorsHuman islet isolation outcomeTemperature control and monitoring during warm digestion and dilution phaseCollection of purified isletsPurified islets and viability assessmentIn vivo assessmentCanine islet isolationPig donor profilesIslet isolation outcome of pig pancreasExperience of preclinical work in the past yearsNTUH New Cell Therapy Center �投影片編號 75ThanksThanks投影片編號 78