A Study on Human Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSCs) Differentiation into Insulin Producing Pancreatic Islet-like Cells

국문 초록 (Abstract)

당뇨병은 만성 고혈당증으로 진단되는 대사질환으로, 인슐린 생산량이 부족하거나 췌장 인슐린 생산 세포가 파괴되어 발생한다. 현재 이 질병을 치료하기 위한 방법은 외부로부터 인슐린을 투여하는 방법이 유일하다. 따라서 당뇨병 환자들은 평생 동안 하루에도 몇 번씩 인슐린을 주사 하여야 하는 어려움이 있다. 이러한 어려움을 최소화하고 삶의 질을 향상시키기 위해 인슐린을 분비하는 β-유사 세포를 이용한 치료방법이 대안일 수 있다. 줄기세포 (인간유도만능 줄기세포: hiPSCs, 배아 줄기세포: hESCs, 중간엽 줄기세포: hMSCs)는 모든 주요 체세포 계통으로 분화할 수 있는 능력으로 인해 인슐린을 생성할 수 있는 아주 매력적인 공급원이다. 와튼 젤리 유래 중간엽 줄기세포는 탯줄의 다른 부분보다 높은 증식률, 다능성, 면역억제 능력과 광범위한 분화 능력을 가지고 있기 때문에 이상적인 공급원이다. 또한 배아줄기세포와 달리 중간엽 줄기세포는 어떠한 윤리적 문제점도 가지고 있지 않다.
본 연구에서는 인슐린을 생성하는 췌장섬 유사세포 분화를 위해 인간 탯줄에서 분리한 와튼 젤리 유래 중간엽 줄기세포를 사용하였다. Chapter I 에서는 와튼 젤리 유래 중간엽 줄기세포의 분리, 중간엽 줄기세포의 특성화 및 계통 분화를 in-vivo에서 검증하였다. Chapter II에서는 와튼 젤리 유래 중간엽 줄기세포를 micro-well, hanging drop, ULA plate 등 다양한 배양 방법을 통하여 3차원 배양하였고, 세포 생존력, 노화, 다계통 분화능, 항염증 또는 면역 조절 인자를 조사하여 2차원 배양된 중배엽성 줄기세포와 비교∙검정하였다. Chapter III 에서는 3차원 배양된 와튼 젤리 유래 중간엽 줄기세포가 인슐린을 생성하는 췌장섬 유사세포로의 분화 능력을 in-vitro에서 평가하고 2차원 배양된 중간엽 줄기세포와 비교∙검정 하였다.
사람 탯줄은 창원 경상국립대학교 병원(GNUCH)에서 출산 후 경상대학교 병원 공인 의료 지침에 따른 동의를 받아 만기 출생아로 부터 공급받았다. 탯줄 조직은 약 2~3 cm 길이의 조각으로 잘라 1% penicillin‐streptomycin이 함유된 DPBS (Dulbecco’s phosphate‐buffered saline)로 여러 번 세척하였다. 이후 동맥, 정맥과 표면 조직이 제거된 와튼 젤리 조직은 작은 조각으로 자른 후 배양 plate에 부착하였다. 부착된 조직은 ADMEM (Advanced Dulbecco’s modified Eagle’s medium)에서 배양되었고, 일주일에 2회 배양액을 교체하였다. 대략 2주내에 중간엽 줄기세포 표현형을 가진 군집형성을 보여주었다.
사람 탯줄 유래 중간엽 줄기세포에서 분리된 와튼 젤리 유래 중간엽 줄기세포는 3번의 계대배양 후 균질한 섬유아세포와 유사한 스핀들 형태를 보였다. 와튼 젤리 유래 중간엽 줄기세포의 표면항원 마커 분석 결과 중배엽성 표면항원인 CD44, CD73, CD105를 발현하는 반면, 음성 항원인 CD34, CD45는 발현하지 않았으며, 중배엽성 계통인 지방세포, 골세포, 연골세포로 분화되었다. 2차원 세포 배양 방법은 세포를 배양하는데 널리 사용되어 왔다. 하지만 2차원 배양은 생체 내 세포의 구조와 미세환경을 완벽히 모방하지 못한다. 3차원 배양을 통한 세포 배양은 생체내 미세환경을 모방하여 광범위하게 응용할 수 있다. 본 연구에서는 3차원 micro-well, hanging drop, ultra-low attachment plate 를 이용한 3차원 배양 방법을 비교하여 와튼 젤리 유래 중간엽 줄기세포의 형태, 생존력, 다능성, 세포 표면 항원 마커, 면역 조절 인자 및 분화 능력에 미치는 영향을 비교하였다. 3차원 배양된 와튼 젤리 유래 중간엽 줄기세포의 형태, 생존력 및 노화는 2차원 배양 세포와 유사하였다. 다능성 마커인 OCT4, SOX2, NANOG의 발현은 2차원배양에 비해 3차원 배양에서 2-8배 증가하였다. 또한 면역조절 인자인 IDO, IL-10, LIF, ANG1, VEGF는 ULA에서 3차원 배양된 와튼 젤리 유래 중간엽 줄기세포에서 유의하게 증가하였다. 3차원 배양에서 지방세포(ADP 및 C/EBP-α), 골세포(OPN 및 RUNX2) 및 definitive endodermal (DE) (SOX17, FOXA2, CXCR4)에 대한 와튼 젤리 유래 중간엽 줄기세포의 분화 능력이 유의하게 향상되었다. 또한 7일, 14일, 28일 동안 2차원 및 3차원 배양에서 와튼 젤리 유래 중간엽 줄기세포의 골세포와 지방세포의 분화 잠재성을 qRT-PCR을 통해 분석하였다. 3차원 배양세포의 분화능력은 7일 및 14일에 크게 증가하였다. 본 연구는 3차원 배양 조건에서 와튼 젤리 유래 중간엽 줄기세포의 줄기세포능이 크게 향상되었고, ULA 배양방법에서 다른 방법보다 높은 다능성 유전자 발현과 향상된 분화 잠재력을 보여주었다.
마지막으로, ULA 배양 방법을 통해 3차원 배양된 와튼 젤리 유래 중간엽 줄기세포를 사용하여 인슐린을 생성하는 췌장섬 유사세포로의 분화를 2차원 배양된 와튼 젤리 유래 중간엽 줄기세포와 비교하였다. 췌장섬 유사세포 분화는 체내의 췌장 발달과정을 모방하여 세포골격 탈중합체를 포함한 저분자를 적합한 시기에 적용하여 6단계로 수행되었다. 또한 돼지 췌장 유래 세포외 매트릭스(pancreatic tissue-derived extracellular matrix)를 사용하여 생체내 미세환경을 제공하였다. 와튼 젤리 유래 중간엽 줄기세포를 definitive endodermal (DE)로 유의하게 구별하는데 가장 적합한 매트릭스 농도는 1 mg/ml로 설정되었다. 또한 인슐린을 생성하는 췌장섬 유사세포 분화를 2차원 배양, ULA배양방법을 통한 3차원 배양, 돼지 췌장 유래 세포외 매트릭스 코팅된 2차원 배양, 돼지 췌장 유래 세포외 매트릭스 코팅된 3차원 배양 등 서로 다른 그룹에서 비교∙분석하였다. 분화된 와튼 젤리 유래 중간엽 줄기세포는 세포 형태에 급격한 변화를 보여주었으며, 섬모양의 세포형태가 관찰되었다. DE와 췌장 전구체의 마커 발현은 2차원 배양에 비해 3차원 배양된 와튼 젤리 중간엽 줄기세포에서 유의하게 높게 발현되었으며, 특히 돼지 췌장 유래 세포외 매트릭스로 코팅된 3차원 배양조건에서 가장 높게 발현되었다. 분화된 세포는 성숙한 β-세포 마커: PDX1, NKX6-1, NGN3, INS, 그리고 ISLET-1을 mRNA 수준에서 qRT-PCR을 통해 발현을 확인하였다. 또한 dithizone으로 염색하여 분화된 세포에서 아연의 생성을 확인하였다. 분화된 세포의 기능을 평가하기 위해 포도당 자극 인슐린 분비분석(GSIS)분석을 수행하였다. 분화된 세포는 포도당의 자극과 인슐린 분비 촉진 환경(저 포도당, 고 포도당 및 염화칼륨)에 의해 인슐린 분비를 확인하였다.
따라서, 본 연구에서 돼지 췌장 유래 세포외 매트릭스 코팅된 3차원 와튼 젤리 유래 중간엽 줄기세포가 in-vitro 환경에서 6단계 분화과정을 통해 인슐린을 생성하는 췌장샘 유사세포를 성공적으로 분화하였다. 향후 연구에서 당뇨병이 유도된 생쥐 모델에서 앞서 분화된 췌장섬 유사세포의 체내 효능을 검증하고자 한다. 또한 3차원 배양조건, 세포외 매트릭스 및 라트룬쿨린 A와 같은 다른 요소들이 세포 운명을 조절하는 신호 메커니즘에 대한 추가 연구를 통해, 췌장 세포를 더 쉽고 저렴하게 분화시킬 수 있는 방법을 밝혀 낼 수 있을 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

The islet of Langerhans of the pancreas contains a majority of insulin-producing cells (70%) i.e. beta cells, which are essential for storing and releasing the insulin hormone to maintain blood glucose level. Both type 1 diabetes mellitus (T1DM), in which the patient's own immune system completely destroys the islets (autoimmune response), and type 2 diabetes mellitus (T2DM), in which patients are unable to respond to insulin because of insulin resistance and insufficient production, can result from abnormalities with the normal functioning of insulin producing cells. The current available strategy to cure the disease is exclusively dependent on the exogenous uptake of insulin. For this, patients need to inject exogenous insulin several times per day throughout their whole lives. To minimize those difficulties and improve their quality of life in the long term, cell therapy using insulin-secreting β-like cells may be one of the best treatments. Stem cells (hiPSCs: human induced pluripotent stem cells, hESCs: human embryonic stem cells, hMSCs: human mesenchymal stem cells) are a very fascinating source of surrogate insulin-producing cells due to their ability to differentiate into all major somatic cell lineages. MSCs from Wharton's jelly (WJ- MSCs) are an ideal source and superior to other parts of the umbilical cord as they have a high proliferation rate, extensive multipotency, hypo-immunogenicity, besides having a wide and efficacious differentiation perspective.
Compared with embryonic stem cells (ESCs), MSCs do not have any ethical issues as ESCs.
In the present study, WJ-MSCs isolated from the full-term human umbilical cord were used for the differentiation of pancreatic insulin-producing islet-like cells. In chapter I, the isolation, in-vitro characterization, and mesenchymal lineage differentiation of WJ-MSCs were studied. In chapter II, we have evaluated the different 3D culturing methods such as 3D micro-well spheroid, HD spheroid, and ULA plate-based spheroid methods for culturing WJ-MSCs. We examined cell viability, senescence, multilineage differentiation potential, anti-inflammatory or immunomodulatory factors of WJ-MSCs in 3D culturing and compared it to 2D cultured cells. Finally, in chapter III, in-vitro trans-differentiation potency of 3D cultured WJ-MSCs into insulin-producing pancreatic islet-like cells was evaluated and compared to that of 2D cultured WJ-MSCs.
Human umbilical cords (UC) were obtained from full-term births at Gyeongsang National University Changwon Hospital (GNUCH) after getting the consent as per authorized medical guidelines set by the GNUH IRB‐2012‐09‐004. UC tissues were cut into pieces of approximately 2-3 cm lengths and rinsed several times with DPBS (Dulbecco’s phosphate‐buffered saline) containing 1% penicillin‐streptomycin. After the removal of two arteries, a vein, and surface tissues, Wharton's jelly (WJ) tissues were chopped into minute pieces with fine scissors and washed with DPBS. Advanced Dulbecco’s modified Eagle’s medium (ADMEM) was added to the dishes after the attachment of ex-planted tissues to the plate surface. The culture dishes were left undisturbed for a few days in the incubator and the media was changed twice a week. Approximately, the cells showed the formation of colonies with an MSCs phenotype within 2 weeks.
The WJ-MSC isolated from the human umbilical cord exhibited adherent fibroblast-like spindle morphology that become homogeneous at passage 3 upon sub-culturing. WJ-MSCs were positive for CD44, CD73, and CD105, whereas negative for CD34 and CD45 marker expression. Further WJ-MSCs were successfully differentiated into mesenchymal lineages (adipocytes, osteocytes, and chondrocytes) in-vitro. The two-dimensional (2D) cell culture method has been widely used to culture cells. However, 2D monolayer culture fails to correctly imitate the architecture and microenvironments of in-vivo cell models. Alternatively, 3D culture may improve the simulations of in-vivo cell microenvironments with wide applications in cell culture and drug discovery. In the present study, we compared various 3D culturing techniques such as 3D micro-well (3D-S), hanging drop (HD), and ultra-low attachment (ULA) plate-based spheroid culture to study their effect on morphology, viability, pluripotency, cell surface markers, immunomodulatory factors, and differentiation capabilities of Wharton’s jelly-mesenchymal stem cells (WJ-MSCs). The cell morphology, viability, and senescence of 3D cultured WJ-MSCs were comparable to cells in 2D culture. The expression of pluripotency markers (OCT4, SOX2, and NANOG) was enhanced up to 2-8 fold in 3D cultured WJ-MSCs when compared to 2D culture. Moreover, the immunomodulatory factors (IDO, IL-10, LIF, ANG1, and VEGF) were significantly elevated in ULA-based 3D cultured WJ-MSCs. Furthermore, significant enhancement in the differentiation potential of WJ-MSCs towards adipocyte (ADP and C/EBP-α), osteocyte (OPN and RUNX2), and definitive endodermal (DE) (SOX17, FOXA2, and CXCR4) lineages in 3D culture conditions were observed. Additionally, the osteogenic and adipogenic differentiation potential of WJ-MSCs in 2D and 3D cultures over the time points 7 days, 14 days, and 28 days was also analyzed using qRT-PCR. The differentiation capacity in 3D cultured groups was significantly increased at 7 days and 14 days. Our study demonstrates that stemness properties of WJ-MSCs were significantly enhanced in 3D culture conditions and ULA-based culture outperformed other methods with high pluripotency gene expression and enhanced differentiation potential.
Finally, ULA-based 3D cultured WJ-MSCs differentiation into insulin-producing pancreatic islet-like cells was evaluated and compared to that of 2D cultured WJ-MSCs. The differentiation was carried out using a six-step pancreatic differentiation protocol through the timed application of a combination of small molecules including a cytoskeletal depolymerizer from WJ-MSCs to enhance the differentiation technique by imitating in-vivo pancreatic development processes. We used pdECM (pancreatic tissue-derived extracellular matrix) to provide a more appropriate microenvironment as in-vivo. pdECM obtained from the porcine pancreas was isolated and characterized. A concentration of 1 mg/ml pdECM was found best to differentiate WJ-MSCs into DE significantly. Further, insulin-producing pancreatic islet-like cell differentiation was performed and comparatively analyzed in different groups such as 2D culture group, ULA-based 3D culture group, pdECM coated 2D cell group, and pdECM coated 3D cultured cell group. The differentiated WJ-MSCs have shown drastic changes in the cell morphology, where islet-like cell morphology has been seen. DE and pancreatic progenitor markers expression were significantly expressed in 3D cultured WJ-MSCs when compared to 2D culture, especially in the pdECM coated 3D culture group. Differentiated cells expressed mature beta-cell markers: PDX1, NKX6-1, NGN3, INS, and ISLET-1 at mRNA and protein levels as evaluated by qRT-PCR and immunocytochemistry, respectively. Moreover, the differentiation was also confirmed by
staining cells with dithizone, which showed the presence of zinc in the differentiated cells. Further, glucose-stimulated insulin secretion (GSIS) assay was performed to evaluate the functionality of differentiated cells. Differentiated cells secreted insulin under the stimulus of glucose and insulin secretion secretagogues (low glucose, high glucose, and KCl).
Therefore, we conclude that pdECM coated 3D cultured WJ-MSCs were successfully differentiated into insulin-producing pancreatic islet-like cells using 6 stage protocol in-vitro. In future studies, we would like to investigate the in-vivo efficacy of differentiated pancreatic islet-like cells in a diabetes model.

목차 (Table of Contents)

• CHAPTER I, Isolation, Characterization and Mesenchymal Lineage Differentiation of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) 30
• ABSTRACT 31
• INTRODUCTION 33
• MATERIALS AND METHODS 36
• Media, chemicals and cell culturing conditions 37
• Isolation and initial culture of human WJ-MSCs 37
• Cell surface antigen profiling of WJ-MSCs 38
• Evaluation of cell proliferation of WJ-MSCs 38
• In-vitro differentiation into Adipocytes, Osteocytes, and Chondrocytes 39
• RNA extraction, cDNA synthesis and qRT-PCR analysis 39
• Immunocytochemistry 41
• Statistical analysis 41
• RESULTS 43
• Basic stem cell characteristics of WJ-MSCs 43
• In-vitro differentiation into mesenchymal lineage 43
• DISCUSSION 49
• REFERENCES 52
• CHAPTER IIComparative analysis of different scaffold-free 3D culture conditions on pluripotency, immunomodulatory factors, and differentiation potential of human Wharton's jelly mesenchymal stem cells 59
• ABSTRACT 60
• INTRODUCTION 62
• MATERIALS AND METHODS 65
• Chemicals and media 65
• Isolation and culture of WJ-MSCs 65
• Cell surface marker expression 66
• 2D and 3D cultivation of WJ-MSCs 66
• Cell recovery and growth characteristics 67
• Senescence-associated β-galactosidase staining 68
• Pluripotency marker expression 68
• Adipogenic differentiation 68
• Osteogenic differentiation 69
• Definitive endoderm differentiation 69
• Immunocytochemistry 69
• RNA extraction, cDNA synthesis, and realtime quantitative PCR (qRT-PCR) 70
• Statistical analysis 71
• RESULTS 74
• Morphology and phenotype expression 74
• Cell viability and senescence 74
• The secretion of immunomodulatory factors under different culture conditions 75
• Pluripotency marker expression 76
• Effect of different culture conditions on the differentiation ability of WJ-MSCs 76
• Adipogenic differentiation of WJ-MSCs 77
• Osteogenic differentiation of WJ-MSCs 78
• Definitive endoderm differentiation of WJ-MSCs 79
• DISCUSSION 86
• REFERENCES 94
• CHAPTER III Differentiation of human Wharton's jelly mesenchymal stem cells into insulin-producing pancreatic islet-like cells 104
• ABSTRACT 104
• INTRODUCTION 107
• MATERIALS AND METHODS 112
• Media, chemicals, and cell culturing conditions 112
• pdECM preparation 112
• Characterization of pdECM 113
• Pancreatic Islet-like cell differentiation 113
• RNA extraction, cDNA synthesis, and realtime quantitative PCR (qRT-PCR) 114
• Immunocytochemistry 115
• Glucose-stimulated insulin secretion (GSIS) 116
• Dithizone (DTZ) staining 116
• Statistical analysis 117
• RESULTS 119
• Development of pancreatic tissue-derived extracellular matrix (pdECM) 119
• In-vitro differentiation of WJ-MSCs into pancreatic endocrine-like cells 119
• DISCUSSION 129
• REFERENCES 136
• ACKNOWLEDGEMENT 143

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