Regenerative potential of human adipose-derived stromal cells of various origins

AbstractIn regenerative concepts, the potential of adult stem cells holds great promise concerning an individualized therapeutic approach. These cells provide renewable progenitor cells to replace aged tissue, and play a significant role in tissue repair and regeneration.In this investigation, the characteristics of different types of adipose tissue are analysed systematically with special attention to their proliferation and differentiation potential concerning the angiogenic and osteogenic lineage. Tissue samples from subcutaneous, visceral, and omental fat were processed according to standard procedures. The cells were characterized and cultivated under suitable conditions for osteogenic and angiogenic cell culture. The development of the different cell cultures as well as their differentiation were analysed morphologically and immunohistochemically from cell passages P1 to P12. Harvesting and isolation of multipotent cells from all three tissue types could be performed reproducibly. The cultivation of these cells under osteogenic conditions led to a morphological and immunohistochemical differentiation; mineralization could be detected. The most stable results were observed for the cells of subcutaneous origin. An osteogenic differentiation from adipose-derived cells from all analysed fatty tissues can be achieved easily and reproducibly. In therapeutic concepts including angiogenic regeneration, adipose-derived cells from subcutaneous tissue provide the optimal cellular base

Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects

Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect..Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects

Investigation of Osteogenic characteristics of human adipose derived stromal cells

Master'sMASTER OF SCIENC

그래핀기반 물질을 이용한 인간 지방유래 기질세포의 연골 분화 효과 연구

학위논문(석사)--서울대학교 대학원 :농업생명과학대학 농생명공학부(바이오모듈레이션전공),2019. 8. 임정묵.Cartilage is a tissue consisting of chondrocytes and extracellular matrix (ECM), which is highly elastic, buffers against a given force, and the friction coefficient of joint cartilage is very low, helping the joints to move in a state of little friction. But cartilage is a kind of expendable body part that wears out as much as it is used, and it is also damaged by inflammation, trauma, and aging. Cartilage has very limited self-renewal because there are no blood vessels, nerves, or lymphatic vessels. Thus, cartilage cannot easily stop when it starts to damage, and damaged cartilage has many limitations about regenerating into cartilage with normal function and structure. Also, the damaged cartilage area becomes more vulnerable to mechanical pressure, so it is easily broken and worn, resulting in larger defects. As such, a number of cartilage-related diseases develop and progresses faster than other tissues. Some typical diseases include degenerative arthritis, which causes pain due to cartilage wear, and others include rheumatoid arthritis, achondroplasia, pyogenic arthritis, and chondrosarcoma. Furthermore, these cartilage-related diseases can lead to bone-related complications if they persist for a long time. These diseases can lead to a reduction in the quality of life and life expectancy of patients, and the loss of substantial medical costs. Therefore, it is very important to regenerate damaged cartilage early, but there are many deficiencies in current cartilage treatment. Although many researchers continue to suggest ways to treat cartilage-related diseases, they are still far from normal and perfect cartilage regeneration. Up until now, studies have been actively conducted to regenerate damaged cartilage tissue using an ideal biomaterial that can mimic and replace cartilage tissue with various cells. Among them, cells that are widely used in the field of regenerative medicine are human adipose-derived stromal cells (hASCs). This cell has a great advantage that it can be easily obtained from any tissue of the human body, and it is suitable as a material for a cell therapy agent because it does not take much time for cell proliferation and can be repeatedly collected. And recently, graphene (G), a carbon-based material, is emerging as a biomaterial in tissue engineering and regenerative medicine. In addition, a variety of graphene-based materials (GBMs) have also been recognized for their research value as biomaterials. GBMs are derivatives of G. Most GBMs are made through the process of oxidation. GBMs that have the advantage of oxidation are more applied in various fields than G. In particular, GBMs play a role as biomaterials due to their various functional groups, biocompatibility, mechanical stability, and other characteristics. And GBMs have been extensively studied in the field of tissue regeneration and repair. However, since G was discovered in 2004 and is a new material only about 20 years old, there is a limit to the lack of prior research related to cartilage. Therefore, cartilage regeneration studies related to GBMs are essential. And because accurate standard indicators for GBMs have not yet been created, characterization and comparison of various GBMs is critical. Based on an understanding of the characteristics of GBMs, this study was conducted to apply to cartilage regeneration studies. The GBMs used in this study were graphene oxide (GO), nano-graphene oxide (nGO) and graphene quantum dot (GQD). All are oxidized GBMs. These are the forms in which parts of the surface have been replaced with oxygen as graphene is oxidized, and the great advantage is that the binding force of each layer of graphite is reduced and distributed well in the solution. Since it can be synthesized in the solution phase, it can be mass-produced and overcome the disadvantages of graphene, which is a very expensive material and has a very high production cost. And because it is easier to attach new functional groups to oxygen than to carbon, it is possible to make more functionalized graphene derivatives. As hydrophobic graphene turns into hydrophilic, oxidized GBMs, affinity with cells increases, and it has the advantage of being easily mixed into culture medium. Therefore, since these advantages allow me to study GBMs as biomaterials, I analyzed the characteristics of each GBMs using Fourier Transform Infra-Red (FT-IR), X-ray Photoelectron Spectroscopy (XPS), and Electrophoretic Light Scattering (ELS). Functional group analysis confirmed that all GBMs were oxidized by showing binding to carboxyl groups, hydroxyl groups and epoxy groups common to surface of GBMs. Through C1s spectra analysis, the degree of oxidation can be determined by the binding and binding ratio of carbon and oxygen. It was found that oxidation is high in the order of nGO, GO, and GQD. Hydrodynamic radius analysis was used to confirm the hydrodynamic radius of each material. This means the radius of the particles in the solution. Therefore, the size of each particle in the solution can be known, and the order in which it is large was confirmed to be GO, nGO and GQD. Finally, Zeta potential analysis was used to confirm the dispersion stability of the particles in solution. The larger the negative value, the higher the dispersion stability. Generally, over –30 means having good stability, so I found that GO and nGO had better dispersion stability than GQD. Therefore, the dispersion stability is high in the order of nGO, GO, and GQD. These data show that all three GBMs are oxidized and help to understand and compare the physical and chemical properties of each particle. Based on the unique properties of these GBMs, I applied them to chondrogenic differentiation of hASCs. Although studies on cartilage regeneration using various cells and biomaterials have been actively studied, there are still no standardized therapies related to cartilage as various problems such as potential side effects on regenerative therapy and verification of effects. So the study was conducted to investigate the effect on the cartilage differentiation of hASCs by applying GBMs, which have recently emerged as biomaterials in the field of regenerative medicine, to cartilage research. First, I conducted a study on the characterization of hASCs. Microscopic photographs showed that hASCs had fibroblastic morphology, and the ability to differentiate into osteocyte, adipocyte and chondrocyte was confirmed through three different dyeing reagents. And based on the GO with the largest particle size, I confirmed the concentration suitable for chondrogenic differentiation of hASCs. As a result, it was confirmed that a concentration of 10 μg / ml or less was suitable. Thus, GO, nGO, and GQD at concentrations of 1 and 10 μg / ml were applied to hASCs. As a result, the size of the chondrocyte pellet was not different from the induction group. However, it was confirmed that Glycosaminoglycans (GAGs) were significantly increased in the 1 μg / ml group of GO and 1 μg / ml of nGO group compared with the induction group through alcian blue staining and toluidine blue staining. These results suggest that GO and nGO, the oxidized graphene, support Chondrogenic differentiation of hASCs. The main purpose of this study was to determine the effect of GBMs on chondrogenic differentiation of hASCs. Particularly, it was confirmed that the effect of chondrogenic differentiation is different according to the particle size and degree of oxidation of GBMs. These studies will help to understand GBMs, which are bio-new materials, and will greatly contribute to the development of new cartilage therapies using these materials.본 논문에서는 3가지의 다른 종류의 그래핀 기반 물질들의 물리적, 화학적 특성을 분석하고 비교를 통해 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점에 대한 이해에 대한 연구와 그래핀 기반 물질들을 생체 소재로 활용한 인간 지방유래 기질세포의 연골 분화에 미치는 영향에 대한 조직 공학 연구를 진행하였다. Chapter 3에서는 그래핀 기반 물질들의 특성을 이해하기 위해 물리적, 화학적 분석에 대한 연구를 진행한 후, 분석된 그래핀 기반 물질들을 이용하여 인간 지방유래 기질세포의 연골 분화에 대한 효과를 확인하였다. 그래핀은 발견된 지 20년이 채 되지 않은 바이오신소재로, 참고할 만한 이전 연구가 많이 없다. 그래서 그래핀 유도체들의 물리, 화학적 표준 지표가 마련되어 있지 않기 때문에 각 각의 특성들을 정확하게 분석하고 이해하는 것이 매우 중요하다. 먼저, Fourier Transform Infra-Red (FT-IR)를 이용하여 각 물질들의 표면에 어떤 작용기들로 구성되어 있는지 확인하였다. 3 종류의 그래핀의 표면에서 공통적으로 카르복실기, 하이드록실기, 에폭시기를 가진다는 것을 확인하였다. 이는 3종류의 그래핀 기반 물질들이 모두 산화되었음을 보여준다. X-ray Photoelectron Spectroscopy (XPS)를 이용해서는 C1s spectra 분석을 하였다. 이를 통해 탄소와 산소의 결합과 비율로 산화 정도를 확인할 수 있었고, 그 결과, 산화 정도는 나노 산화 그래핀, 산화 그래핀, 그래핀 양자점 순서로 큰 것을 확인하였다. Electrophoretic light scattering (ELS)를 이용해서는 각 물질들의 유체역학적 반경을 측정하였다. 이는 용액 속에서 입자가 영향을 주는 반경을 의미하기 때문에, 입자의 크기를 알 수 있다. 그 결과, 입자의 크기는 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점 순서로 큰 것을 확인하였다. 마지막으로, ELS를 이용하여 zeta potential 값을 측정하였다. 제타 포텐셜 값이 높은 음의 값을 가질수록 용액에서의 분산 안정성이 높으며, 일반적으로 –40이 넘어가면 안정성이 좋다는 것을 의미한다. 그 결과, 분산 안정성이 높은 순서는 나노 산화 그래핀, 산화 그래핀, 그래핀 양자점 순서임을 확인하였다. 이러한 결과들을 통해 그래핀 기반 물질들의 표면 작용기, 산화 정도, 입자의 크기, 분산안정성에 대해 이해할 수 있었고, 이는 그래핀 기반 물질들을 다양한 분야에서 적용시킬 때 많은 도움을 줄 것으로 생각한다. 그리고 분석한 그래핀 기반 물질들을 바이오소재로서 이용하여 인간 지방유래 기질세포의 연골 분화에 미치는 영향에 관한 연골 재생 연구를 하였다. 소수성인 그래핀을 산화시켜 만든 그래핀 기반 물질들은 친수성으로 성질이 바뀌면서 세포와의 친화성이나 배양 배지에 쉽게 분산될 수 있는 장점을 가져 조직공학 분야에 접목시키기 유리하다. 그래서 나는 먼저, 인간 지방유래 기질세포의 형태학적인 분석과 골, 지방, 연골로의 분화능에 대한 분석을 하였다. 인간 지방유래 기질세포는 섬유아세포와 같은 모양을 가졌으며, 골, 지방, 연골로의 분화능을 가지는 것으로 확인하였다. 그리고 나서 인간 지방유래 기질세포의 연골 분화에 적합한 산화 그래핀의 농도를 확인하였다. 산화 그래핀을 기준으로 한 이유는 입자의 크기가 가장 크고, 조직 공학과 관련된 참고문헌이 가장 많기 때문이다. 그 결과, 10 ㎍/㎖의 이하의 농도에서 적합한 것을 확인하였다. 그래서 1과 10 ㎍/㎖의 농도의 산화 그래핀, 나노 산화 그래핀, 그래핀 양자점을 이용해 인간 지방유래 기질세포의 연골 분화 영향을 확인하였을 때, 콘드로사이트의 펠렛 크기에는 영향을 주지 않았다. 하지만 alcian blue staining과 toluidine blue staining으로 조직학적 분석을 하였을 때, 1 ㎍/㎖의 농도의 산화 그래핀과 나노 산화 그래핀 군에서 양성대조군에 비해 GAGs의 함량이 유의적으로 증가함을 확인하였다. 그 결과, 100 nm 이상의 크기를 가진 산화 그래핀 기반 물질들이 인간 지방유래 기질세포의 연골분화에 효과가 있음을 확인하였다. 그러므로 이 연구를 통해 재생 의학이나 조직공학 분야에서 떠오르는 바이오 신소재로 자리매김을 할 수 있는 가능성을 확인하였다. 앞으로 그래핀 기반 물질들이 세포와 세포 외 기질 등 주변 환경에 미치는 영향이나 세포 독성, 적합한 배양 조건에 관한 깊이 있는 연구가 더 진행된다면 충분히 조직 공학적 응용이 가능해 질 것이다PART I : GENERAL INTRODUCTION & LITERATURE REVIEW 1 CHAPTER 1 : General Introduction 2 CHAPTER 2 : Literature Review 9 1. Cartilage 10 1.1. Definition 10 1.2. Composition 10 1.3. Cartilage-related diseases 15 2. Tissue engineering 22 2.1. Definition 22 2.2. Materials . 22 2.2.1 Cells . 23 2.2.2 Biomaterials . 26 3. Cartilage regeneration 29 PART II : EFFECT OF GRAPHENE-BASED MATERIALS ON CHONDROGENIC DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED STROMAL CELLS 31 CHAPTER 1 : Effect of graphene-based materials on chondrogenic differentiation of human adipose-derived stromal cells 32 1. Introduction 33 2. Materials and Methods 39 3. Results 44 4. Discussion 58 PART III : GENERAL DISCUSSION & CONCLUSION 61 1. General discussion and conclusion 62 REFERENCES 66 SUMMARY IN KOREAN 95Maste

Transplantation of Adipose Derived Stromal Cells into the Developing Mouse Eye

Adipose derived stromal cells (ADSCs) were transplanted into a developing mouse eye to investigate the influence of a developing host micro environment on integration and differentiation. Green fluorescent protein-expressing ADSCs were transplanted by intraocular injections. The age of the mouse was in the range of 1 to 10 days postnatal (PN). Survival dates ranged from 7 to 28 post transplantation (DPT), at which time immunohistochemistry was performed. The transplanted ADSCs displayed some morphological differentiations in the host eye. Some cells expressed microtubule associated protein 2 (marker for mature neuron), or glial fibrillary acid protein (marker for glial cell). In addition, some cells integrated into the ganglion cell layer. The integration and differentiation of the transplanted ADSCs in the 5 and 10 PN 7 DPT were better than in the host eye the other age ranges. This study was aimed at demonstrating how the age of host micro environment would influence the differentiation and integration of the transplanted ADSCs. However, it was found that the integration and differentiation into the developing retina were very limited when compared with other stem cells, such as murine brain progenitor cell

Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency: The role of DREAM in the maintenance of hESCs

Little is knownabout the functions of downstreamregulatory element antagonist modulator (DREAM) inembryonic stem cells (ESCs). However, DREAM interacts with cAMP response element-binding protein (CREB) in a Ca2+-dependent manner, preventing CREB binding protein (CBP) recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAMdepletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs) function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs), human adipose-derived stromal cells (hASCs), human bone marrow-derived stromal cells (hBMSCs), and human newborn foreskin fibroblasts (hFFs), and that transitory inhibition of DREAMin hESCs reduces their pluripotency, increasing differentiation.We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAMacts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.Junta de Andalucía, Consejería de Innovación Ciencia y Empresa, FEDER CTS-6505; INP-2011- 1615-900000; P10-CVI-6095Instituto de Salud Carlos III, FEDER RD12/0019/0028; PI10/00964; PI14/0101

Human Adipose Stromal Cells Increase Survival and Mesenteric Perfusion Following Intestinal Ischemia and Reperfusion Injury

OBJECTIVE: Intestinal ischemia can quickly escalate to bowel necrosis and perforation. Transplantation of stem cells presents a novel treatment modality for this problem. We hypothesized that: human adipose-derived stromal cells (hASCs) would increase survival and mesenteric perfusion to a greater degree compared with differentiated cellular controls following ischemic intestinal injury, and improved outcomes with hASC therapy would be associated with preservation of intestinal histological and tight junction architecture, and lower levels of systemic inflammation following intestinal injury. METHODS: hASCs and keratinocytes (differentiated cellular control) were cultured on polystyrene flasks at 37°C in 5% CO2 in air. Adult male C57Bl6J mice were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root identified, and intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 min with a noncrushing vascular clamp. Following ischemia, the clamp was removed, and the intestines were returned to the abdominal cavity. Before abdominal closure, 2 million hASCs or keratinocytes in 250 μL of phosphate-buffered saline (carrier for cells and control solution) were infused into the peritoneum. Animals were allowed to recover for 12 or 24 h (perfusion, histology, cytokine, and immunofluoresence studies), or 7 days (survival studies). Intestinal perfusion was assessed by laser Doppler imaging. Intestinal tissue segments were stained with hematoxylin and eosin, as well as antibodies for the tight junction protein claudin-1. Separate aliquots of intestine, liver, and lung tissue were homogenized and assessed for inflammatory cytokines via multiplex beaded assay. RESULTS: Animals administered hASCs following intestinal ischemia and reperfusion (I/R) injury had significantly greater 7-day survival and better postischemic recovery of mesenteric perfusion compared with vehicle or keratinocyte therapy. hASCs also abated intestinal mucosal destruction, facilitated preservation of intestinal tight junctions, and decreased the systemic inflammatory response to injury. CONCLUSIONS: Human adipose-derived stromal cells improved survival and mesenteric perfusion and attenuated the mucosal damage associated with intestinal I/R injury. hASCs should be considered as a plausible cell source for novel cellular treatment plans following intestinal ischemia

Fabrication And Characterization Of Thiol-Acrylate Based Polymer for Bone Tissue Engineering Application

Thiol-acrylate materials have been demonstrated to have therapeutic potential as biocompatible scaffolds for bone tissue regeneration due to their osteoconductivity, biodegradability, and well-suited mechanical properties. This study connects the mechanical properties and stability of thiol-acrylate polymer with cell adhesion and proliferation of human adipose derived stromal cells. The polymer presented in this study, trimethylolpropane ethoxylate triacrylate-co-trimethylolpropane tris (3-mercaptopropionate) (TMPeTA-co-TMPTMP), was synthesized by an amine-catalyzed Michael addition reaction. Physical, mechanical, and chemical characterizations were performed on the polymeric scaffold, followed by preliminary in vitro cytocompatibility tests. Live/dead staining assays showed significant differences in cell adhesion for TMPeTA (692 and 912 MW). Collectively, these results highlight the potential for these thiol-acrylate based polymers to be a versatile, biocompatible scaffold for bone tissue engineering applications

Simvastatin coating of TiO2 scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells

AbstractBone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO2) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO2 scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering