이 연구는 NIH/NIAMS K01AR071512 및 CIRM DISC0-14350에서 Dmitriy Sheyn에 의해 부분적으로 지원되었습니다. 두 개의 렌티바이러스 패키징 플라스미드는 Simon Knott 실험실(Cedars-Sinai Medical Center, Department of Biomedical Sciences)에서 기증한 것입니다.

효과적인 세포 기반 치료를 위한 iPSC 유래 iTenocyte 활용

<ol>
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모든 저자는 공개할 이해 상충이 없습니다.

이 프로토콜에서 iTenocyte는 (1) iMSC에 대한 iPSC의 유도, (2) 렌티바이러스 벡터를 사용한 SCX의 과발현, (3) 2D 단축 장력을 통한 세포 성숙의 세 가지 주요 단계를 통해 생성됩니다.
iPSC를 iMSC로 구별하기 위해 제시된 프로토콜은 이전에 그룹2에서 설명했습니다. 이 발표 이후, 임상시험에서 iMSC를 사용하기 위한 확립된 프로토콜(21,22,23)<su...

힘줄과 인대 복구의 현대 문제를 해결하기 위해서는 세포 기반 치료에 적합한 적절한 세포 후보가 필요합니다. 힘줄 복구를 위한 조직 공학의 한 가지 연구 방법은 골수 유래 중간엽 기질 세포(BM-MSC) 및 지방 조직 유래 기질 세포(ASC)를 잠재적 전략으로 탐색하는 것입니다. 이 세포는 다능 능력, 매우 풍부하며 생체 내에서 재생 잠재력을 가지고 있습니다. 또한 동물 모델에서 향상된 치유 능력과 향상된 기능적 결과를 보여주었습니다1. 그럼에도 불구하고 이러한 세포는 제한된 자가 재생 능력, 표현형 다양성, 특히 힘줄 형성 능력이 제한되어 있습니다. 유도만능줄기세포(iPSC) 기술은 뛰어난 자가 재생 능력과 타의 추종을 불허하는 발달 적응성으로 인해 이러한 제약에 대한 솔루션을 제공합니다. 우리 연구팀과 다른 연구팀은 iPSC를 중간엽 기질 세포 유사 개체(iMSC)로 성공적으로 분화시켰습니다2,3. 따라서 iMSC는 힘줄 세포 치료 응용 분야를 위한 동종 공급원이 될 수 있는 잠재력을 가지고 있습니다.
공막(SCX)은 힘줄 발달에 필수적인 전사 인자이며 분화된 건세포에 대해 가장 먼저 검출할 수 있는 마커로 간주됩니다. 또한 SCX는 유형 1a1 사슬 콜라겐 1(COL1a1), 모호크(MKX) 및 테노모듈린(TNMD)을 포함한 다운스트림 힘줄 분화 마커를 활성화합니다 4,5,6. 힘줄 성숙 중에 발현되는 다른 유전자로는 튜불린 중합 촉진 단백질 패밀리 멤버 3(TPPP3) 및 혈소판 유래 성장 인자 수용체 알파(PDGFRa)7가 있습니다. 이러한 유전자는 힘줄의 발달과 성숙에 필수적이지만, 불행히도 힘줄 조직에만 국한된 것은 아니며 뼈나 연골과 같은 다른 근골격계 조직에서 발현됩니다 5,7.
힘줄 발달 중 마커의 발현 외에도 기계 자극은 배아 힘줄 발달 및 치유에 필수적인 요소입니다 4,5,6. 힘줄은 기계적으로 반응하며 성장 패턴은 환경에 따라 변화합니다. 분자 수준에서, 생체역학적 단서는 건세포의 발달, 성숙, 유지 및 치유 반응에 영향을 미친다8. 다양한 바이오리액터 시스템이 생리학적 부하와 생체역학적 단서를 모델링하는 데 활용되었습니다. 이러한 모델 시스템 중 일부에는 생체 외 조직 로딩, 이축 또는 단축 장력을 적용하는 2D 세포 로딩 시스템, 스캐폴드 및 하이드로겔을 사용하는 3D 시스템이 포함됩니다 9,10. 2D 시스템은 힘줄 특이적 유전자 또는 세포 운명의 맥락에서 세포의 형태에 대한 기계적 자극의 효과를 연구할 때 유리한 반면, 3D 시스템은 세포-ECM 상호 작용을 보다 정확하게 복제할 수 있습니다 9,10.
2D 로딩 시스템에서는 세포와 배양 기질 사이의 변형률이 균일하며, 이는 세포의 세포골격에 가해지는 하중을 완전히 제어할 수 있음을 의미합니다. 이축 로딩(bi-axial loading)과 비교하여, 일축 로딩(uniaxial loading)은 생리학적으로 더 관련성이 높은데, 이는 텐노사이트(tenocyte)가 생체 내 콜라겐 다발(collagen bundle)로부터 일축 로딩(uniaxial loading)을 주로 받기 때문이다 9. 일상 활동 중에 힘줄은 최대 6%의 변형률(11)까지 단축 인장 하중을 받는 것으로 나타났다. 특히, 이전 연구에서는 4%-5%의 생리학적 범위 내에서 로딩이 SCX 및 TNMD와 같은 힘줄 관련 마커 발현을 보존하고 콜라겐 생산을 증가시켜 테노겐 분화를 촉진하는 것으로 나타났습니다 9,10. 10% 이상의 균주는 외상적으로는 관련이 있지만 생리적으로는 관련이 없을 수 있다12,13.
여기에서는 건세포 생성에 필수적일 수 있는 기계적 및 생물학적 자극의 시너지 효과를 고려한 프로토콜이 제시됩니다. 먼저 유세포 분석을 사용하여 MSC 표면 마커로 확인된 배아체를 성장 인자에 단기간 노출 시켜 iPSC를 iMSC로 유도하는 재현 가능한 방법을 설명합니다. 그런 다음 SCX(iMSCSCX+)의 안정적인 과발현을 갖도록 iMSC를 엔지니어링하는 렌티바이러스 형질도입 방법을 자세히 설명합니다. 추가 세포 성숙을 위해 iMSCSCX+ 는 피브로넥틴 코팅 실리콘 플레이트에 파종되고 CellScale MCFX 바이오리액터를 사용하여 최적화된 단축 장력 프로토콜을 거칩니다. tenogenic 잠재력은 이른 힘줄 마커와 늦은 힘줄 마커의 상향 조절과 콜라겐 침착을 관찰함으로써 확인되었다14. 이 iTenocytes 생성 방법은 힘줄 세포 치료 응용 분야를 위한 무제한 기성품 동종 소스를 제공할 수 있는 개념 증명입니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>StemCell Technologies</td>
			<td>7920</td>
			<td>cell dissociation reagent</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic solution</td>
			<td>Thermofisher</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD105</td>
			<td>Ancell</td>
			<td>326-050</td>
			<td></td>
		</tr>
		<tr>
			<td>APC mouse anti-human CD44</td>
			<td>BD Biosciences</td>
			<td>559942</td>
			<td></td>
		</tr>
		<tr>
			<td>APC mouse IgG2 K isotype control</td>
			<td>BD Biosciences</td>
			<td>555745</td>
			<td></td>
		</tr>
		<tr>
			<td>BenchMark fetal bovine serum</td>
			<td>GeminiBio</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Biglycan</td>
			<td>Thermofisher</td>
			<td>Hs00959143_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A3733</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type I alpha 1 chain human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs00164004_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen type III alpha 1 chain human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs00943809_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Millipore Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td>
			<td>Thermofisher</td>
			<td>11054020</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#39;s minimum essential medium (EMEM)</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD90</td>
			<td>BD Biosciences</td>
			<td>555595</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG1-PE</td>
			<td>Bio-Rad</td>
			<td>STAR117</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293T/17</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM, no phenol red</td>
			<td>Thermofisher</td>
			<td>21056023</td>
			<td></td>
		</tr>
		<tr>
			<td>iPSCs: 83i-cntr-33n1</td>
			<td>Cedars-Sinai iPSC Core Facility</td>
			<td>N/A</td>
			<td>https://biomanufacturing.cedars-sinai.org/product/cs83ictr-33nxx/</td>
		</tr>
		<tr>
			<td>Isotype Control Antibody, mouse IgG2a-FITC</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-271</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut serum replacement</td>
			<td>Thermofisher</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermofisher</td>
			<td>2503081</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>basement membrane matrix</td>
		</tr>
		<tr>
			<td>MechanoCulture FX</td>
			<td>CellScale</td>
			<td>N/A</td>
			<td>stretching apparatus</td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids solution</td>
			<td>Thermofisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mohawk human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs00543190_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>StemCell Technologies</td>
			<td>100-0276</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermofisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet-derived growth factor receptor A human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs00998018_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(2-hydroxyethyl methacrylate)</td>
			<td>Sigma Aldrich</td>
			<td>192066</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene infection/transfection reagents</td>
			<td>Millipore Sigma</td>
			<td>TR-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human&#160; TGF-beta 1 protein human Taqman primer</td>
			<td>RnD Systems</td>
			<td>240-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Scleraxis human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs03054634_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>SCXA (SCX) (NM_00108050514) human tagged ORF clone</td>
			<td>OriGene</td>
			<td>RC224305L4</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone plates</td>
			<td>CellScale</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Millipore Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tenascin C human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs00370384_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tenomodulin human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs00223332_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombospondin 4 human Taqman primer</td>
			<td>Thermofisher</td>
			<td>Hs00170261_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection reagent, BioT</td>
			<td>Bioland Scientific LLC</td>
			<td>B01-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermofisher</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin polymerization promoting protein family member 3</td>
			<td>Thermofisher</td>
			<td>Hs03043892_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Biogems</td>
			<td>1293823</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cell-derived itenocytes via combined scleraxis overexpression and 2d uniaxial tension

힘줄과 인대 복구에 대한 오늘날의 과제는 힘줄 재생을 촉진하기 위한 세포 기반 치료에 적합하고 효과적인 후보를 식별해야 합니다. 중간엽 기질 세포(MSC)는 힘줄 복구를 위한 잠재적인 조직 공학 전략으로 연구되어 왔습니다. 그들은 다능하고 생체 내에서 재생 잠재력을 가지고 있지만 자체 재생 능력이 제한되어 있으며 표현형 이질성을 나타냅니다. 유도만능줄기세포(iPSC)는 높은 자가 재생 능력과 탁월한 발달 가소성으로 인해 이러한 한계를 우회할 수 있습니다. 건세포 발달에서 공막(Scx)은 힘줄 분화의 중요한 직접 분자 조절자입니다. 또한 기계 조절은 배아 힘줄의 발달과 치유를 이끄는 핵심 요소인 것으로 나타났습니다. 따라서 우리는 힘줄 생성에 필수적일 수 있는 생물학적 및 기계적 자극의 시너지 효과를 캡슐화하는 프로토콜을 개발했습니다. iPSC는 중간엽 기질 세포(iMSC)가 되도록 유도되었으며 유세포 분석을 통해 고전적인 중간엽 기질 세포 마커로 특성화되었습니다. 다음으로, 렌티바이러스 벡터를 사용하여 iMSC를 안정적으로 과발현하도록 형질주입했습니다(iMSCSCX+). 이러한 iMSCSCX+ 세포는 2D 바이오리액터를 사용하여 단축 인장 하중을 통해 iTenocyte로 더 성숙할 수 있습니다. 결과 세포는 초기 및 후기 힘줄 마커의 상향 조절과 콜라겐 침착을 관찰하는 것이 특징이었습니다. 이 iTenocytes 생성 방법은 힘줄 세포 치료 응용 분야를 위한 잠재적으로 무제한의 기성품 동종 세포 소스를 개발하는 연구원을 지원하는 데 사용할 수 있습니다.

To tackle the contemporary issues in tendon and ligament repair, there&#39;s a requirement for a pertinent cell candidate suitable for cell-based therapies. One avenue of investigation in tissue engineering for tendon repair involves the exploration of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and adipose tissue-derived stromal cells (ASCs) as potential strategies. These cells have multipotent capability, great abundance, and regenerative potential in vivo. Additionally, they have shown enhanced healing capacity and improved functional outcomes in animal models1. Nonetheless, these cells exhibit restricted self-renewal capabilities, phenotypic diversity, and notably, limited capacity for tendon formation. Induced pluripotent stem cell (iPSC) technology offers a solution to these constraints due to its remarkable self-renewal capacity and unmatched developmental adaptability. Our research team and others have achieved successful differentiation of iPSCs into mesenchymal stromal cell-like entities (iMSCs)2,3. As such, iMSCs have the potential to be an allogenic source for tendon cell therapy applications.
Scleraxis (SCX) is a transcription factor essential for tendon development and is considered the earliest detectable marker for differentiated tenocytes. Additionally, SCX activates downstream tendon differentiation markers, including type 1a1 chain collagen 1 (COL1a1), mohawk (MKX), and tenomodulin (TNMD), among others4,5,6. Other genes expressed during tendon maturation include tubulin polymerization-promoting protein family member 3 (TPPP3) and platelet-derived growth factor receptor alpha (PDGFRa)7. While these genes are essential for tendon development and maturation, they are unfortunately not unique to tendon tissue and are expressed in other musculoskeletal tissues like bone or cartilage5,7.
In addition to the expression of markers during tendon development, mechanostimulation is an essential element for embryonic tendon development and healing4,5,6. Tendons are mechanoresponsive, and their growth patterns change in response to their environment. At the molecular level, biomechanical cues affect the development, maturation, maintenance, and healing responses of tenocytes8. Various bioreactor systems have been utilized to model physiological loads and biomechanical cues. Some of these model systems include ex vivo tissue loading, 2D cell loading systems applying bi-axial or uniaxial tension, and 3D systems using scaffolds and hydrogels9,10. 2D systems are advantageous when studying the mechanical stimulation&#39;s effects on either tendon-specific genes or the morphology of the cells in the context of cell fate, while 3D systems can more accurately replicate cell-ECM interactions9,10.
In 2D loading systems, the strain between the cells and the culture substrate is homogeneous, meaning that the applied load on the cytoskeleton of the cells can be fully controlled. In comparison to bi-axial loading, uniaxial loading is more physiologically relevant, as tenocytes are predominantly subjected to uniaxial loading from collagen bundles in vivo9. It is found that during daily activities, tendons are subjected to uniaxial tensile loading up to 6% strain11. Specifically, previous studies have found that loading within the physiological ranges of 4%-5% has been shown to promote tenogenic differentiation by preserving tendon-related marker expression like SCX and TNMD, as well as increased collagen production9,10. Strains of more than 10% may be traumatically relevant but not physiologically relevant12,13.
Here, a protocol is presented that takes into account the synergistic effect of mechanical and biological stimulation that may be essential for the generation of tenocytes. We first describe a reproducible method to induce iPSCs into iMSCs via short-term exposure of embryoid bodies to growth factors, confirmed by MSC surface markers using flow cytometry. We then detail a lentiviral transduction method to engineer iMSCs to have stable overexpression of SCX (iMSCSCX+). For further cell maturation, the iMSCSCX+ are seeded into fibronectin-coated silicone plates and undergo an optimized uniaxial tension protocol using CellScale MCFX bioreactor. The tenogenic potential was confirmed by observing the upregulation of early and late tendon markers, as well as collagen deposition14. This method of generating iTenocytes is a proof-of-concept that may offer an unlimited off-the-shelf, allogeneic source for tendon cell therapy applications.

저자 스포트라이트: 힘줄 복구를 위한 세포 및 조직 공학의 발전 

Generation of induced pluripotent stem cell-derived iTenocytes via combined scleraxis overexpression and 2D Uniaxial tension(공막 과발현 및 2D 단축 장력 결합을 통한 유도만능줄기세포 유래 iTenocytes 생성)

Tendon injury is one of the most prevalent musculoskeletal problems in the US, affecting an estimated 33 million people annually. Our research focuses on developing a cell and tissue engineering approach for repairing tendons that can drastically improve patient outcomes. Current technologies used to advance tendon research include the use of stem cells from different sources like induced pluripotent stem cells in combination with the use of bioreactors, genetic engineering biomaterials, and other tissue engineering techniques to mimic the environmental cues needed for tendon.Our understanding of how tendons develop from their origins is limited. There are several well-known genes that are very highly expressed during tendon maturation. However, they're unfortunately not unique to the tendon tissue.This makes it difficult for researchers to standardly assess successful tendon differentiation. A protocol takes advantage of remarkable IPC differentiation potential and what we know so far on tendon development. Our goal is to develop an allogeneic cell source for tendon cell therapy applications.Our laboratories focused on elucidating the pathways involved with tendon sulfate to better understand tendon development. We want to implement this knowledge in combination with tissue engineering technologies and biomaterials to develop more effective alternatives for traditional tendon repair.

인간 iPSC와 iMSC의 분화 앞서 설명한 바와 같이, iPSC를 iMSC로 분화하기 위한 현재의 프로토콜은 배아체(embryoid body)의 형성을 포함한다(2). 이 과정은 iPSC에서 iMSC를 유도하는 데 약 10일이 걸립니다(그림 1A). 그러나 새로 생성된 iMSC를 최소 두 번 통과하는 것이 좋습니다. 이는 젤라틴 코팅 플레이트의 필요성을 없애는 데 도움이 될 뿐만 아니라 안...

Watch this Scientific Journal Video about Advancements in Cell and Tissue Engineering for Tendon Repair at JoVE.com

이 논문에서는 렌티바이러스 벡터를 사용하여 공막의 과발현과 2D 바이오리액터를 통한 단축 스트레칭이 결합된 iPSC 유래 중간엽 기질 세포를 생성하여 iTenocyte를 생성하는 절차에 대해 설명합니다.

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to ...

힘줄 부상은 미국에서 가장 흔한 근골격계 문제 중 하나로, 매년 약 3,300만 명에게 영향을 미칩니다. 우리의 연구는 환자의 결과를 크게 개선할 수 있는 힘줄 복구를 위한 세포 및 조직 공학 접근 방식을 개발하는 데 중점을 둡니다. 힘줄 연구를 발전시키는 데 사용되는 현재 기술에는 힘줄에 필요한 환경 신호를 모방하기 위해 생물 반응기, 유전 공학 생체 재료 및 기타 조직 공학 기술의 사용과 함께 유도 만능 줄기 세포와 같은 다양한 출처의 줄기 세포 사용이 포함됩니다.힘줄이 그 기원에서 어떻게 발달하는지에 대한 우리의 이해는 제한적입니다. 힘줄 성숙 중에 매우 높게 발현되는 잘 알려진 몇 가지 유전자가 있습니다. 그러나 불행히도 힘줄 조직에만 있는 것은 아니다.이로 인해 연구자들은 성공적인 힘줄 분화를 표준적으로 평가하기가 어렵습니다. 프로토콜은 놀라운 IPC 차별화 잠재력과 힘줄 발달에 대해 지금까지 알고 있는 것을 활용합니다. 우리의 목표는 힘줄 세포 치료 응용 분야를 위한 동종 세포 소스를 개발하는 것입니다.우리 연구실은 힘줄 발달을 더 잘 이해하기 위해 황산힘과 관련된 경로를 설명하는 데 중점을 두었습니다. 우리는 이 지식을 조직 공학 기술 및 생체 재료와 결합하여 구현하여 전통적인 힘줄 복구를 위한 보다 효과적인 대안을 개발하고자 합니다.

generation-induced-pluripotent-stem-cell-derived-itenocytes-via

Research

JoVE Journal

Bioengineering

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

1.2K 조회수.  Cedars-Sinai Medical Center. 힘줄 부상은 미국에서 가장 흔한 근골격계 문제 중 하나로, 매년 약 3,300만 명에게 영향을 미칩니다. 우리의 연구는 환자의 결과를 크게 개선할 수 있는 힘줄 복구를 위한 세포 및 조직 공학 접근 방식을 개발하는 데 중점을 둡니다. 힘줄 연구를 발전시키는 데 사용되는 현재 기술에는 힘줄에 필요한 환경 신호를 모방하기 위해 생물 반응기, 유전 공학 생체 재료 및 기타 조직 ?...

비디오: 저자 스포트라이트: 힘줄 복구를 위한 세포 및 조직 공학의 발전 

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D bioreactor.