세포의 재생 --＞ 물리적 자극(운동)에 의해서 활성화된다. Nature review

[문형철]

mechanobiology of stem cells

간단하게 정리하면

치료적 운동(기계적 힘)이 어떻게 세포를 재생하는가? 

viscoelestic 조직

--> 자극 

--> 반응... 

--> 세포 재생...

이 논문에 의하면 인체 조직은 기계적 힘에 영향을 받는 세포로 구성되어 있다. 이 기계적 힘이 세포를 재생을 유도한다. 이렇게 말하고 있어요.

tensile(장력), compressive(압박력), shear force(전단력)... 이런 힘들이 주어져야 기계적 이온채널이 열리고 세포가 재생된다. 이런 그림

Nat Rev Mol Cell Biol. Author manuscript; available in PMC 2018 Jun 1.

Published in final edited form as:

Nat Rev Mol Cell Biol. 2017 Dec; 18(12): 728–742.

Published online 2017 Nov 8. doi: 10.1038/nrm.2017.108

PMCID: PMC5803560

NIHMSID: NIHMS936567

PMID: 29115301

Mechanical forces direct stem cell behaviour in development and regeneration

Kyle H. Vining and David J. Mooney*

Author information Copyright and License information PMC Disclaimer

The publisher's final edited version of this article is available at Nat Rev Mol Cell Biol

Associated DataSupplementary Materials

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Abstract

Stem cells and their local microenvironment, or niche, communicate through mechanical, cues to regulate cell fate and cell behaviour, and to guide developmental processes. During embryonic development, mechanical forces are involved in patterning and organogenesis.

The physical environment of pluripotent stem cells regulates their differentiation and self-renewal. Mechanical and physical cues are also important in adult tissues, where adult stem cells require physical interactions with the extracellular matrix to maintain their potency. In vitro, synthetic models of the stem cell niche can be used to precisely control and manipulate the biophysical and biochemical properties of the stem cell microenvironment and examine how the mode and magnitude of mechanical cues, such as matrix stiffness or applied forces, direct stem cell differentiation and function.

Fundamental insights on the mechanobiology of stem cells also inform the design of artificial niches to support stem cells for regenerative therapies.

요약

줄기세포와 그 국소 미세 환경, 

즉 틈새는 기계적 신호를 통해 소통하여 

세포의 운명과 세포 행동을 조절하고 발달 과정을 안내합니다. 

배아 발달 과정에서 기계적 힘은 패터닝과 기관 형성에 관여합니다. 

만능 줄기세포의 물리적 환경은 

세포의 분화와 자기 재생을 조절합니다. 

The physical environment of pluripotent stem cells regulates their differentiation and self-renewal.

기계적 및 물리적 단서는 성체 조직에서도 중요한데, 

성체 줄기세포는 그 효능을 유지하기 위해 세포 외 기질과의 

물리적 상호 작용이 필요합니다. 

Mechanical and physical cues are also important in adult tissues, where adult stem cells require physical interactions with the extracellular matrix to maintain their potency

시험관 내에서 

줄기세포 틈새 stem cell niche 의 합성 모델을 사용하여 

줄기세포 미세 환경의 생물물리학적 및 생화학적 특성을 

정밀하게 제어 및 조작하고 

매트릭스 강성 또는 

가해진 힘과 같은 기계적 단서의 모드와 크기가 

줄기세포 분화 및 기능을 어떻게 유도하는지 조사할 수 있습니다. 

mode and magnitude of mechanical cues,

such as matrix stiffness or applied forces

줄기세포의 기계생물학에 대한 근본적인 통찰력은 

재생 치료를 위한 줄기세포를 지원하기 위한

artificial niches 를 설계하는 데도 도움이 됩니다.

1. Introduction

Forces are generated and resisted across many magnitudes and length scales in biology, from a sub-cellular level, for example by actomyosin motors to an organismal level, such as in response to gravity. Similar to intrinsic and extrinsic biochemical factors, mechanical cues resulting from both intracellularly-generated and externally-applied forces have broad impact on stem cell function. Mechanical interactions mediated by adhesion to the extracellular matrix (ECM) and cell-cell junctions play a key part in transmitting forces to and between cells, which regulate intracellular signalling pathways (FIG. 1).

생물학에서 힘은 액토미오신 모터와 같은 세포 이하 수준부터 중력에 대한 반응과 같은 유기체 수준에 이르기까지 다양한 크기와 길이에 걸쳐 생성되고 저항됩니다. 내재적 및 외재적 생화학적 요인과 마찬가지로, 세포 내에서 생성되는 힘과 외부에서 가해지는 힘으로 인한 기계적 단서는 줄기세포 기능에 광범위한 영향을 미칩니다.

세포 외 기질(ECM)과 세포 간 접합에 의해 매개되는

기계적 상호 작용은

세포 내 신호 경로를 조절하는

세포 간 힘 전달에 중요한 역할을 합니다(그림 1).

Figure 1

Stem cells exert forces and are subject to external forces, which regulate their intracellular signaling pathways. A) Intrinsic, or cell-generated forces, (Fi) are generated intracellularly and transferred to other cells through cell-cell junctions, like cadherin receptors, or via traction on extracellular matrix (ECM) adhesion ligands that are bound to integrin receptors. Cells are directly coupled by cell-cell junctions, which link the intrinsic forces of one cell to the cytoskeleton of another. Indirect mechanical coupling between cells occurs by intrinsic forces exerted on an ECM, to which two or more cells are adhered. Physical properties, e.g., elastic modulus, E, of the ECM, govern how mechanical cues are transduced. Extrinsic forces (Fe) are externally applied by shear or tension/compression on cells, and can be sensed by mechanically-gated ion channels, changes in receptor-ligand binding, deformation of the cytoskeleton, and the primary cilium. The cytoskeleton generates and transfers forces from membrane proteins to intracellular structures, like the nucleus. B) ECM and intracellular pathways are biochemically coupled by mechanotransduction pathways. Matrix mechanical resistance to intrinsic forces regulates the stability of focal adhesion complexes that contain focal adhesion kinase (FAK), which phosphorylates and activates mechano-responsive signaling elements, such as mitogen-associated protein kinase (MAPK) and Rho kinase (RhoA). RhoA regulates mechanical feedback by activating ROCK, which phosphorylates myosin light chain (MLP) to generate actomyosin forces. For example, RhoA phosphorylates ROCK, which activates non-muscle myosin II contractility by phosphorylation of myosin light chain (MLP), and upregulates mechano-transduction pathways, such as MAL, a G-actin-binding coactivator of serum response factor (SRF),88.89 Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ),90 which induce nuclear transcription via activation and nuclear translocation. Nuclear signaling pathways, such as serum response factor (SRF) and myocardin-related transcription factor-A (MRFTA), regulate transcription of mechanoresponsive genes. For example, mechanics-dependent expression‎ of miR-21 regulates fibrogenic behavior. Wnt activation also promotes activation and nuclear translocation of YAP/TAZ and beta-catenin.160 Mechanical forces are also physically directly coupled to the nucleus via lamina proteins, such as lamin A (LMNA), which can affect chromatin structure and epigenetic regulation of transcription.

줄기세포는 힘을 발휘하고 세포 내 신호 전달 경로를 조절하는 외부 힘의 영향을 받습니다.

A) 내재적 힘 또는 세포 생성 힘(Fi)은 세포 내에서 생성되어 카데린 수용체와 같은 세포-세포 접합을 통해 또는 인테그린 수용체에 결합된 세포 외 기질(ECM) 접착 리간드의 견인을 통해 다른 세포로 전달됩니다. 세포는 한 세포의 고유한 힘을 다른 세포의 세포 골격에 연결하는 세포-세포 접합에 의해 직접 결합됩니다. 세포 간의 간접적인 기계적 결합은 두 개 이상의 세포가 부착된 ECM에 가해지는 고유한 힘에 의해 발생합니다. ECM의 탄성 계수 E와 같은 물리적 특성은 기계적 신호가 전달되는 방식을 결정합니다. 외력(Fe)은 세포에 전단 또는 장력/압박에 의해 외부에서 가해지는 힘으로, 기계적으로 게이트된 이온 채널, 수용체-리간드 결합의 변화, 세포 골격의 변형 및 일차 섬모에 의해 감지될 수 있습니다. 세포 골격은 막 단백질에서 핵과 같은 세포 내 구조로 힘을 생성하고 전달합니다.

B) ECM과 세포 내 경로는 기계 전달 경로에 의해 생화학적으로 결합됩니다. 내재적 힘에 대한 매트릭스의 기계적 저항은 미토겐 관련 단백질 키나아제(MAPK) 및 Rho 키나아제(RhoA)와 같은 기계 반응 신호 요소를 인산화하고 활성화하는 국소 접착 키나아제(FAK)를 포함하는 국소 접착 복합체의 안정성을 조절합니다. RhoA는 미오신 경쇄(MLP)를 인산화하여 액토미오신 힘을 생성하는 ROCK를 활성화하여 기계적 피드백을 조절합니다. 예를 들어, RhoA는 미오신 경쇄(MLP)의 인산화를 통해 비근육 미오신 II 수축성을 활성화하는 ROCK를 인산화하고, 활성화 및 핵 전위를 통해 핵 전사를 유도하는 혈청 반응 인자(SRF),88.89 예 관련 단백질(YAP), PDZ 결합 모티프를 가진 전사 활성화제(TAZ)90 등의 기계 전달 경로를 상향 조절하는 역할을 합니다. 혈청 반응 인자(SRF) 및 심근 관련 전사인자-A(MRFTA)와 같은 핵 신호 경로는 기계 반응성 유전자의 전사를 조절합니다. 예를 들어, miR-21의 기계 의존적 발현은 섬유화 작용을 조절합니다. Wnt 활성화는 또한 YAP/TAZ 및 베타카테닌의 활성화와 핵 전위를 촉진합니다.160 기계적 힘은 라민 A(LMNA)와 같은 라미나 단백질을 통해 핵에 물리적으로 직접 결합되어 염색질 구조와 후성유전적 전사 조절에 영향을 줄 수 있습니다.

기계 민감성 채널 (MSC), 기계 민감성 이온 채널 또는 스트레치 게이트 이온 채널은 광범위한 외부 기계적 자극에 대해 기계적 스트레스에 반응할 수 있는 막 단백질입니다. [1][2][3][4] 박테리아, 고세균, 진핵생물 등 세 가지 생명 영역의 유기체 막에 존재하며[5] 촉각, 청각, 균형 감각을 비롯한 여러 시스템의 센서일 뿐만 아니라 심혈관 조절과 삼투성 항상성(예: 갈증)에도 관여합니다. 채널은 박테리아에서 음이온과 양이온을 모두 통과하는 비선택적 채널부터 진핵생물에서 Ca2+, K+ 및 Na+를 통과시키는 양이온 선택적 채널, 박테리아와 진핵생물에서 매우 선택적인 K+ 채널까지 침투하는 이온에 대한 선택성이 다양합니다.모든 유기체와 모든 세포 유형은 기계적 자극을 감지하고 이에 반응합니다.[6] MSC는 외부 또는 내부[7] 자극에 대한 반응으로 전기 및 이온 유속 신호를 모두 생성할 수 있는 기계 변환기 역할을 합니다.[8] 박테리아의 극심한 장력 하에서 MSCL 및 MSCS와 같은 비선택적 MSC는 용해를 방지하기 위한 안전 밸브 역할을 합니다. 고등 생물의 특수 세포에서는 다른 유형의 MSC가 청각과 촉각의 기초가 되며 근육 조정에 필요한 스트레스를 감지합니다. 그러나 이러한 채널 중 어느 것도 복제되지 않았습니다. 또한 MSC는 중력을 감지하여 식물이 위와 아래를 구분할 수 있게 해줍니다. MSC는 압력에는 민감하지 않지만 국소 스트레스, 즉 주변 지질 이중층의 장력에 민감합니다.[9]

Cells exert intrinsic forces on their environment, that is, on the ECM and neighbouring cells, through various mechanisms, including actomyosin contractility and cytoskeletal assembly. Conversely, cell-extrinsic shear, tensile and compressive forces are applied on stem cells from an external load. Whether generated intrinsically or extrinsically, the impact that mechanical forces have on cell behaviour depends on the biological context of a cell, such as biochemical cues and epigenetic state, and on various aspects of its physical environment. The microenvironment may be composed of fluids, solids, gases, or other cells, and provides external resistance or compliance that may store or dissipate forces (Boxes 12). Owing to the complexity of these mechanical interactions, it is important to develop systems in which each type of mechanical cue can be controlled and de-coupled from others to understand how they affect stem cells.

세포는

액토미오신 수축성 및 세포 골격 조립을 포함한

다양한 메커니즘을 통해 환경,

즉 ECM 및 주변 세포에 고유한 힘을 가합니다.

반대로

세포 외적 전단, 인장 및 압축력은

외부 하중으로부터 줄기세포에 가해지는 힘입니다.

cell-extrinsic shear, tensile and compressive forces are applied on stem cells from an external load.

내재적으로 발생하든 외재적으로 발생하든

기계적 힘이

세포 행동에 미치는 영향은

생화학적 단서 및 후성유전학적 상태와 같은

세포의 생물학적 맥락과 물리적 환경의 다양한 측면에 따라 달라집니다.

미세 환경은

액체, 고체, 기체 또는 기타 세포로 구성될 수 있으며,

힘을 저장하거나 방출할 수 있는 외부 저항 또는 순응도를 제공합니다(상자12).

이러한 기계적 상호 작용의 복잡성으로 인해

각 유형의 기계적 단서를 제어하고

다른 단서와 분리하여 줄기세포에 미치는 영향을 이해할 수 있는 시스템을 개발하는 것이 중요합니다.

Box 1

Introduction to mechanical terms and concepts

Various concepts and terms are used to describe and quantify the types and magnitudes of mechanical properties, and the relationship between forces and deformations.

•  
•  

The relationship between the force and deformation, or stress and strain, in a system is dependent on the properties of the material that transfers the stress/strain (e.g., the matrix to which cells apply tractional forces). If a material effectively stores energy during the transfer, the material is termed to be Elastic. In linearly elastic materials there is a linear relationship between stress and strain, and energy (e.g., forces) applied to the material are stored (e.g., deformations induced by cells protruding into an elastic material will be stored in the material and “push” back against the cell). For example, rubbers and covalently-crosslinked hydrogels are typically considered to be elastic, and their rigidity or stiffness is determined by the modulus (e.g., elastic modulus, shear modulus), which is closely related to the density of crosslinks between the polymer chains comprising the network. The elasticity, or Young’s modulus, of an elastic material, can be approximated in linearly elastic solids by the slope of the stress versus strain curve at small strains (typically 1–5%).

Many tissues, cells, and extracellular matrices combine mechanical properties of solids and liquids, and these types of materials are termed viscoelastic. They behave as elastic solids and as viscous fluids simultaneously. Viscous fluids demonstrate flow, or permanent deformation, in response to applied forces, and are described by their viscosity, which relates the rate and extent of flow to an applied force. Viscoelastic materials will exhibit stress relaxation (decrease in the stress required over time to maintain a constant level of strain), and creep (increase in strain over time in response to a constant stress). Viscoelastic materials can dissipate applied loads via their permanent deformation, allowing for cells to remodel viscoelastic matrices even in the absence of degradation, and without a build-up in the material of forces that “push” back on the cell.

The term stiffness is typically used in mechanobiology as a metric of the rigidity of a matrix as sensed by cells via application of cell-generated forces. A stiffer matrix will require higher forces to deform the network, whereas a softer matrix can be deformed by lower forces. The stiffness of matrices is often determined under the assumption of linear elastic behavior, making it synonymous with elasticity.

기계적 용어 및 개념 소개

기계적 특성의 유형과 크기, 힘과 변형 사이의 관계를 설명하고 정량화하기 위해 다양한 개념과 용어가 사용됩니다.

• 응력stress은 물체에 가해지는 힘의 크기를 나타내는 것으로, 힘이 가해지는 면적에 정규화된 값입니다. 응력은 힘/면적, σ = F/A로 계산되며 일반적으로 뉴턴/㎡ 또는 파스칼 단위로 표시됩니다.
• 스트레인은 가해진 힘으로 인한 변형의 척도로, 물체 길이의 정규화된 변화로 표시됩니다. 스트레인(ε) = 현재 길이/초기 길이-1 = Δl/l0-1이며 일반적으로 무차원 분수 또는 백분율로 표시됩니다.

시스템에서 힘과 변형 또는 응력과 변형 사이의 관계는 응력/변형을 전달하는 재료의 특성(예: 셀이 견인력을 가하는 매트릭스)에 따라 달라집니다. 전달 과정에서 에너지를 효과적으로 저장하는 소재를 탄성이 있는 소재라고 합니다. 선형 탄성 재료 linearly elastic materials 에서는 응력과 변형률 사이에 선형 관계가 있으며, 재료에 가해지는 에너지(예: 힘)가 저장됩니다(예: 탄성 재료로 돌출된 세포에 의해 유도된 변형은 재료에 저장되어 세포에 다시 “밀려” 되돌아오게 됩니다). 예를 들어, 고무와 공유 결합 하이드로겔은 일반적으로 탄성이 있는 것으로 간주되며, 그 강성 또는 강성은 네트워크를 구성하는 폴리머 사슬 사이의 가교 밀도와 밀접한 관련이 있는 계수(예: 탄성 계수, 전단 계수)에 의해 결정됩니다. 탄성 재료의 탄성 또는 영 계수는 선형 탄성 고체에서 작은 변형률(일반적으로 1-5%)에서 응력 대 변형률 곡선의 기울기로 근사화할 수 있습니다.

많은 조직, 세포 및 세포 외 매트릭스는

고체와 액체의 기계적 특성을 결합하고 있으며,

이러한 유형의 물질을 점탄성  viscoelastic 이라고 합니다.

점탄성 물질은

탄성 고체와 점성 유체처럼 동시에 작동합니다.

점성 유체는 가해진 힘에 반응하여 흐름 또는 영구적인 변형을 나타내며, 점도는 가해진 힘에 대한 흐름의 속도와 정도를 나타내는 점도로 설명할 수 있습니다. 점탄성 소재는 응력 완화(일정한 수준의 변형을 유지하기 위해 시간이 지남에 따라 필요한 응력의 감소)와 크리프(일정한 응력에 대한 반응으로 시간이 지남에 따라 변형이 증가)를 나타냅니다. 점탄성 소재는 영구적인 변형을 통해 가해지는 하중을 분산시킬 수 있으므로 성능이 저하되지 않고 셀을 '밀어내는' 힘이 소재에 축적되지 않은 상태에서도 셀이 점탄성 매트릭스를 리모델링할 수 있습니다.

The term stiffness is typically used in mechanobiology as a metric of the rigidity of a matrix as sensed by cells via application of cell-generated forces.

강성stiffness이라는 용어는 일반적으로 기계생물학에서 세포가 생성한 힘을 가하여 세포가 감지한 매트릭스의 강성을 측정하는 지표로 사용됩니다. 매트릭스가 단단할수록 네트워크를 변형시키는 데 더 큰 힘이 필요하지만, 매트릭스가 부드러울수록 더 낮은 힘으로도 변형시킬 수 있습니다. 매트릭스의 강성은 종종 선형 탄성 거동을 가정하여 결정되며, 이는 탄성과 동의어로 사용됩니다.

Box 2

Mechanical properties of tissues, cells, and matrix

Elastic and viscoelastic properties of tissues, cells, and matrix are typically measured by mechanical tests in which a known and defined stress or strain is applied on a sample while the other is measured. Methods of rheology, the study of flow and deformation of matter, are used to characterize both the elastic (G′, storage modulus) and viscous (G″, loss modulus) behavior of viscoelastic materials by dynamically controlling the rate and amount of strain or stress while measuring the other. Bulk compression or tension measurements are used to measure the elastic modulus (E, Young’s modulus), which relates to the density of crosslinks in a hydrogel, by applying a uniaxial strain and measuring the stress. These uniaxial compression measurements or shear compression measurements can also be used to measure the stress relaxation or creep of a material, which measures the time-dependent changes in stress or strain resulting from the application of a constant level of strain or stress. Methods such as atomic force microscopy, nanoindentation, optical tweezers, force traction microscopy, and micro-aspiration, probe the stiffness at the nano- and micro-scale, which allows for mapping of mechanical topographies or gradients in materials. Representative mechanical properties of select tissues and materials are provided in Table 1.

조직, 세포 및 매트릭스의 기계적 특성

조직, 세포 및 매트릭스의 탄성 및 점탄성 특성

Elastic and viscoelastic properties of tissues, cells, and matrix은

일반적으로 시료에 알려진 정의된 응력 또는 변형이 가해지는 동안

다른 하나는 측정하는 기계적 테스트를 통해 측정됩니다.

물질의 흐름과 변형에 대한 연구인 유변학 방법은 변형률 또는 응력의 속도와 양을 동적으로 제어하면서 다른 하나를 측정하여 점탄성 물질의 탄성(G′, 저장 계수) 및 점성(G″, 손실 계수) 거동을 모두 특성화하는 데 사용됩니다. 벌크 압축 또는 장력 측정은 일축 변형을 가하고 응력을 측정하여 하이드로겔의 가교 밀도와 관련된 탄성 계수(E, 영의 계수)를 측정하는 데 사용됩니다. 이러한 일축 압축 측정 또는 전단 압축 측정은 재료의 응력 이완 또는 크리프를 측정하는 데에도 사용할 수 있으며, 이는 일정한 수준의 변형 또는 응력을 가하여 발생하는 응력 또는 변형의 시간 의존적 변화를 측정합니다. 원자력 현미경, 나노 압입, 광학 핀셋, 힘 견인 현미경, 미세 흡인 등의 방법은 나노 및 마이크로 스케일에서 강성을 조사하여 재료의 기계적 지형 또는 경사도를 매핑할 수 있습니다. 일부 조직 및 재료의 대표적인 기계적 특성은 표 1에 나와 있습니다.

Table 1

Representative mechanical properties of tissue and materials

Tissue Type       Tissue or MaterialTypical Applications                                                                                      E (kPa)t1/2 (s)

Interstitial and connective	Fat	-	0.02147	10047
	Tendon	-	310,000147	1149
	Skin	-	4.5–8148	-
Vascular	Carotid artery	-	90147	-
Nervous	Spinal cord	-	27–89147	-
	Brain	-	0.2–1.0147,150	10047
Viscera	Lung	-	5147	-
	Kidney	-	2.5147	-
	Liver	-	0.64147	10047
	Lymph node	-	0.12147	-
	Mammary gland	-	0.16147	-
Musculoskeletal	Cardiac muscle	-	20–150147	-
	Skeletal muscle	-	10–100147,150	-
	Pre-calcified bone	-	30150	-
	Bone marrow	-	0.3–24.7151	1047
	Cartilage	-	20150	-
	Articular cartilage	-	950147	-
	Bone - Cancellous	-	350,000152	-
	Bone – Compact	-	11,500,000152	-
	Tooth dentin	-	>10,000,000153	-
	Tooth enamel	-	~100,000,000153	-
Embryonic	Gastrulation	-	0.01154	-
Natural ECM	Collagen hydrogels	-	0.01–6155,158	1155
	Fibrin hydrogels	-	0.01–0.5155	1155
	rBM (Matrigel)	-	0.01–0.5155	50155
	Gelatin – covalently crosslinked	-	0.6–13156	n/a
	Hyaluronic acid hydrogels	-	4–9545	n/a
Synthetic matrix	Alginate hydrogels	2D and 3D matrices, programmable viscoelastic properties, coupled with cell adhesive ligands or interpenetrating network with natural ECM, option for degradability	0.1–11044,47	44–330047
	Polyacrylamide	2D elastic substrates coated with ECM	0.1–4054	n/a
	Agarose hydrogels	2D elastic substrates coated with ECM	5–10044,155	>1000155
	Poly(ethylene glycol) hydrogels	Covalently-crosslinked 2D and 3D matrices, coupled with cell adhesive ligands, incorporation of degradable elements	0.1–16044,110	n/a
	Polystyrene	Traditional tissue culture material (plasma-treated for promoting cell adhesion)	3,000,000125	n/a
	PDMS	2D elastomer for applying extrinsic strain	5–100115	n/a
	PDMS micropillars	2D studies to measure and/or manipulate cell intrinsic forces	2.8–60157*	n/a

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Values = XReference, E = Elastic modulus (assume E = 2G′ (1+ ν), G′ storage modulus, ν = 0.5), t1/2 = time to reach half of initial normalized stress during constant applied strain (Box 1), rBM = recombinant basement membrane, PDMS = polydimethylsiloxane; - indicates value not reported

*Effective elastic modulus, Eeff

Understanding how mechanical forces regulate stem cell behaviour provides key insights into the understanding of developmental biology, and for the development of regenerative therapies. In this Review, we provide an overview of the types of mechanical cues that affect stem cells and of biomaterial-based systems that can be used to control and manipulate these cues. We focus on how different types of forces regulate stem cell behaviours in early development and organogenesis, control stem cell fate, including differentiation and self-renewal, and can be exploited to promote regeneration.

기계적 힘이 

줄기세포의 행동을 어떻게 조절하는지를 이해하는 것은 

발달 생물학을 이해하고 

재생 치료법을 개발하는 데 중요한 통찰력을 제공합니다. 

이 리뷰에서는 

줄기세포에 영향을 미치는 기계적 신호의 유형과 

이러한 신호를 제어하고 조작하는 데 사용할 수 있는 생체 재료 기반 시스템에 대한

 개요를 제공합니다. 

또한 

다양한 유형의 힘이 초기 발달과 기관 형성에서 

줄기세포의 행동을 조절하고 

분화 및 자기 재생을 포함한 줄기세포의 운명을 제어하며 

재생을 촉진하는 데 어떻게 활용될 수 있는지에 초점을 맞춥니다.

2. Mechanical cues guide development

The growth, differentiation and morphogenesis of a developing embryo is dependent on intrinsic and extrinsic mechanical forces that drive the assembly of cells and promote growth into higher order structures (Table S1).1 Cell-cell adhesion transmits tensile forces and, as embryonic development progresses, cells become mechanically coupled to matrix proteins in tissues by adhesion molecules (FIG. 2A), which helps drive morphogenesis and maintain stem cells’ position and fate in their niche. This mechanical coupling enables storage of information over time. For example, changes in ECM induced by cells early in development can mechanically trigger changes in interacting cells at a later stage. Moreover, mechanical cues can be more rapidly propagated over long distances during morphogenesis than biochemical cues (for example, the transmission of forces in highly elastic substrates like elastin is almost instantaneous).

2. 발달을 안내하는 기계적 단서

발달 중인 배아의 성장, 분화 및 형태 형성은

세포의 조립을 유도하고 고차 구조로의 성장을 촉진하는

내재적 및 외재적 기계적 힘에 의존합니다(표 S1).1

세포 간 접착은

인장력을 전달하고

배아 발달이 진행됨에 따라 세포는

접착 분자에 의해 조직의 기질 단백질에 기계적으로 결합되어

형태 형성을 촉진하고

줄기세포의 위치와 운명을 유지하는 데 도움을 줍니다(그림 2A).

이러한

기계적 결합을 통해

시간이 지남에 따라 정보를

저장할 수 있습니다.

예를 들어, 발달 초기에 세포에 의해 유도된 ECM의 변화는 이후 단계에서 상호 작용하는 세포의 변화를 기계적으로 촉발할 수 있습니다. 또한 기계적 신호는 생화학적 신호보다 형태 형성 과정에서 장거리에 걸쳐 더 빠르게 전파될 수 있습니다(예: 엘라스틴과 같이 탄성이 높은 기질에서 힘의 전달은 거의 즉각적으로 이루어집니다).

Figure 2

Mechanobiology in the developmental niche.

A) While development progresses, intrinsic forces exerted by cells transition from largely cell-cell to more cell-matrix transmission as matrix content in tissues increases. B) Higher astral tension (white triangles) on the posterior axis (right side, bold arrow) of dividing cells is generated by cortical tension and microtubule polymerization, which results in asymmetry in cell size after division.8 C) Cell-cell intrinsic forces in early development modify the pattern of embryonic epithelial adhesion and intercalation, which results in elongation of the anterior- (A) posterior (P) axis.4 D) During epithelial branching morphogenesis of the fetal submandibular salivary gland, cells exert intrinsic actomyosin contractility and traction forces on the extracellular matrix (ECM) (red), which assembles at a clefting region and promotes cell proliferation (pink arrow) in the budding region.33 ECM contains domains of heparan sulfate (HS) that bind FGF growth factors to promote epithelial bud elongation by differentially increasing their local concentration.159 Thus, concerted biochemical and mechanical cues work together to generate proper organ form.

발달 틈새 분야의 기계생물학.

A) 발달이 진행되는 동안 세포가 가하는 내재적 힘은 조직의 매트릭스 함량이 증가함에 따라 주로 세포-세포 간 전달에서 세포-매트릭스 간 전달로 전환됩니다.

B) 분열하는 세포의 후축(오른쪽, 굵은 화살표)에서 더 높은 천상 장력(흰색 삼각형)은 피질 장력과 미세소관 중합에 의해 생성되어 분열 후 세포 크기의 비대칭을 초래합니다.8

C) 발달 초기 세포-세포 고유력은 배아 상피 부착 및 상호 연화의 패턴을 수정하여 전방(A) 후방(P) 축의 신장을 초래합니다.4

D) 태아 턱밑 침샘의 상피 분지 형태 형성 과정에서 세포는 고유한 액토미오신 수축성과 견인력을 세포 외 기질(ECM)에 발휘하여(빨간색), 갈라진 부위에 모이고 발아 부위에서 세포 증식을 촉진합니다(분홍색 화살표).33 ECM에는 헤파란 설페이트(HS) 도메인이 포함되어 있는데, 이 도메인은 FGF 성장 인자와 결합하여 국소 농도를 차별적으로 증가시켜 상피 싹의 신장을 촉진합니다.159 따라서 생화학적 및 기계적 신호가 함께 작용하여 적절한 장기 형태를 생성합니다.

Intrinsic and extrinsic forces guide early embryo development

Prior to implantation, self-organization of the embryo and specification of the germ layers into a blastocyst rely on contractile mechanical cues.2 Tensions are relieved as the embryo transitions from a spherical to elongated body form3 and an intercalated pattern of cell-cell epithelial junctions is generated.4 The presence of intrinsic cell-generated forces in development was demonstrated by observing the deformation of embryonic tissues following their macro-scale dissection at different stages of morphogenesis,5 as well as nano-dissection of the actomyosin network at apical cell junctions. The latter showed that anisotropy of intrinsic forces is sufficient to promote elongation of embryonic epithelia.6

Intrinsic mechanical tensile and compressive forces that regulate multiple aspects of embryonic morphogenesis are generated by non-muscle myosin II in actomyosin complexes6. For example, actomyosin forces promote the establishment of an anterior-posterior axis of development by promoting asymmetric spindle positioning (FIG. 2B)7,8 and the remodelling of intercalated epithelial junctions4 (FIG. 2C). Axis elongation depends on tension generated by multicellular cables of actomyosin9 with both elastic and viscous mechanical behaviour (Box 1), as demonstrated by laser-severing.10 A catch-bond mechanism, in which tensile forces promote the stabilization of cadherin-catenin complexes [G] bound to actin filaments, demonstrates how these cell-cell receptor interactions communicate biochemical signals through mechanical forces.11 These forces also control gastrulation by regulating epithelial invagination12–14 and progenitor cell sorting within the germ layers15, as well as dorsal closure [G].16 Intrinsic forces in embryonic tissues can also be observed when embryonic stem cells (ES cells) are dissociated, as they also display cortical tension [G] generated by actomyosin contractility. This is required to reduce apoptosis, but inhibition of Rho-associated protein kinase (ROCK) [G] relieves this dependence.17

In addition to being subjected to intrinsic mechanical forces, the embryo receives external mechanical input from the fluid surrounding its intercalated cell layers that induce patterning. Fluid shear forces during early development regulate left-versus-right body asymmetry by signalling through primary cilium and by generating morphogen gradients.18,19 Morphogens Sonic hedgehog (SHH) and retinoic acid (RA) are secreted in ‘nodal vesicular parcels’ (NVPs) in a FGF-dependent manner and transported to the left by the nodal flow.19 However, important questions on the mechanobiology of embryonic morphogenesis remain largely unaddressed, such as how mechanical forces interplay with genetic and epigenetic changes, which could inform approaches to manipulate intrinsic forces by control of transcription, and how robust pattern formation results even in the face of natural variations in the magnitude and duration of intrinsic and extrinsic forces.

내재적 및 외재적 힘이 초기 배아 발달을 유도합니다.

착상 전, 배아의 자기 조직화와 배반포로 배아 층의 구체화는 수축성 기계적 신호에 의존합니다.2 배아가 구형에서 길쭉한 체형으로 전환되면서 긴장이 완화되고3 세포-세포 상피 접합의 상호 결합 패턴이 생성됩니다.4 형태 형성의 여러 단계에서 거시적 규모의 해부를 통해 배아 조직의 변형을관찰하고5 정단 세포 접합부에서 액토미오신 네트워크의 나노 해부를 통해 발달에 내재된 세포 생성력의 존재를 입증했습니다. 후자는 내재적 힘의 이방성이 배아 상피의 신장을 촉진하기에 충분하다는 것을 보여주었습니다.6

배아 형태 형성의 여러 측면을 조절하는 내재적 기계적 인장 및 압축력은 액토미오신 복합체에서 비근육 미오신 II에 의해 생성됩니다6. 예를 들어, 액토미오신 힘은 비대칭 스핀들 포지셔닝(그림 2B)7,8과 상피 접합부의 리모델링4을 촉진하여 전방-후방 발달 축의 형성을 촉진합니다(그림 2C). 축 신장은 레이저 절단으로 입증된 바와 같이 탄성 및 점성 기계적 거동을 모두 가진 액토미오신9의 다세포 케이블에 의해 생성된 장력에 의존합니다(상자 1).10 인장력이 액틴 필라멘트에 결합된 카데린-카테닌 복합체[G]의 안정화를 촉진하는 캐치본드 메커니즘은 이러한 세포-세포 수용체 상호작용이 기계적 힘을 통해 생화학 신호를 전달하는 방법을 보여 줍니다.11 이러한 힘은 또한 상피 침윤12-14 및 생식층 내 전구 세포 분류15와 등쪽 폐쇄[G]를 조절하여 위장 조절을 제어합니다.16 배아 줄기 세포(ES 세포)가 해리될 때 배아 조직의 내재적 힘도 관찰할 수 있는데, 이는 액토미오신 수축성에 의해 생성되는 피질 긴장[G]을 나타내기 때문입니다. 이는 세포 사멸을 줄이는 데 필요하지만, Rho 관련 단백질 키나아제(ROCK)[G]를 억제하면 이러한 의존성이 완화됩니다.17

배아는 내재적인 기계적 힘을 받는 것 외에도, 세포 간 층을 둘러싼 유체로부터 패턴화를 유도하는 외부 기계적 입력을 받습니다. 초기 발달 중 유체 전단력은 일차 섬모를 통한 신호와 모포겐 구배를 생성하여 왼쪽 대 오른쪽 신체 비대칭을 조절합니다.18,19 모포겐 소닉 헤지혹(SHH)과 레티노산(RA)은 FGF 의존적인 방식으로 '결절 소포(NVP)'에서 분비되고 결절 흐름에 의해 왼쪽으로 운반됩니다.19 그러나 배아 형태 형성의 기계 생물학에 대한 중요한 질문, 즉 기계적 힘이 유전적 및 후성 유전적 변화와 어떻게 상호 작용하는지, 전사 조절을 통해 내재적 힘을 조작하는 접근법을 알려줄 수 있는지, 내재적 및 외재적 힘의 크기와 기간의 자연적 변화에도 불구하고 강력한 패턴 형성이 어떻게 발생하는지 등은 대부분 해결되지 않은 채로 남아 있습니다.

Cell-ECM interactions guide later stages of development

Early in development cell-cell contacts predominate, but as development progresses, progenitor cells differentiate and begin to deposit ECM to which they adhere20 (FIG. 2A). The number of collagen fibrils and fiber length steadily increase in embryonic development, whereas collagen content remains stable in post-natal growth.21

Cell intrinsic forces are transmitted to matrix proteins such as fibronectin by integrin cell surface receptors, which can form focal complexes that generate forces of 1–3 nN/μm2.22 Increased integrin expression‎ in tissues may alter how cells respond to ECM stiffness, in part by stabilizing matrix-integrin interactions.23 Thus, as the matrix content of tissues increases during development, the coupling increases between ECM mechanical properties and intrinsic mechanical forces, one aspect of mechanosensing.

Cell-ECM interactions enable biological systems to contextualize stimuli because cells respond differently to the same mechanical stimulus depending on their micro-mechanical or biochemical environment. In embryonic avian skin, mechanical and biochemical factors are coupled to the positioning and patterning of hair follicles. Mechanical resistance to dermal cell contraction upregulates β-catenin activation, which drives downstream follicular gene expression‎ programs, including bone morphogenic protein-2 (BMP2).24 Furthermore, mechanics direct multi-scale developmental processes by connecting macro-scale physical inputs with nano-scale molecular signals through chemical cues.25 For example, force transmission through specific receptors enables generic mechanical signals to be transduced into specific cell responses based on the type of integrin receptors that are expressed in the cells or ligands present in the ECM. Logically, mechanical transduction needs to be carefully controlled to maintain homeostasis, because mechanical stimuli are often not specific. Moreover, ECM provides a mechanical rheostat for cells in four-dimensions – time & space, because the matrix can dissipate elastic energy and change over time by stiffening, degradation and matrix deposition.

Mechanical forces regulate cell fate decisions during organogenesis as progenitor cells are directed to diverse specialized functions in fetal organs. The process of germ band extension generates mechanical forces that promote differentiation of the stomodeum [G] and midgut tissues by inducing the expression‎ of Twist, which is one of the earliest expressed embryonic patterning genes.26 Tensile forces, arising from stretching of bronchial epithelium during intrauterine breathing, support development of smooth muscle in the lung.27 Shear forces generated by maternal blood flow promote fetal hematopoiesis and the morphogenesis of cardiac tissues28–30, and signal to epithelia in the developing kidney via the primary cilia, which require polycystin-1 and polycystin-2 proteins to promote kidney morphogenesis.31 In limb development, stress, strain, hydrostatic pressure, and fluid flow precede regional ossification and subsequent bone collar formation.32 The underlying molecular mechanisms are the subject of current studies. These examples highlight how organ development is affected by the physical nature of their microenvironment. Research aiming to recapitulate these developmental processes from stem cells in vitro will require sophisticated systems in which forces can be tightly controlled.

세포-ECM 상호 작용은 발달의 후기 단계를 안내합니다.
발달 초기에는 세포와 세포의 접촉이 우세하지만 발달이 진행됨에 따라 전구세포가 분화하여 ECM을 침착하기 시작합니다20 (그림 2A). 콜라겐 원 섬유의 수와 섬유 길이는 배아 발달 과정에서 꾸준히 증가하는 반면, 출생 후 성장에서는 콜라겐 함량이 안정적으로 유지됩니다.21

세포 고유 힘은 인테그린 세포 표면 수용체에 의해 피브로넥틴과 같은 기질 단백질로 전달되며, 이는 1-3 nN/μm2의 힘을 생성하는 초점 복합체를 형성할 수 있습니다.22 조직에서 인테그린 발현이 증가하면 부분적으로 기질-인테그린 상호작용을 안정화하여 세포가 ECM 강성에 반응하는 방식을 변경할 수 있습니다.23 따라서 발달 중에 조직의 기질 함량이 증가하면 기계 감지의 한 측면인 ECM 기계적 특성과 고유 기계적 힘 사이의 결합이 커집니다.
세

포는 미세 기계적 또는 생화학적 환경에 따라 동일한 기계적 자극에 다르게 반응하기 때문에 세포-ECM 상호 작용을 통해 생물학적 시스템이 자극을 맥락화할 수 있습니다. 배아 조류 피부에서는 기계적 및 생화학적 요인이 모낭의 위치 및 패턴 형성에 결합되어 있습니다. 진피 세포 수축에 대한 기계적 저항은 β-카테닌 활성화를 상향 조절하여 골형성 단백질-2(BMP2)를 포함한 모낭 유전자 발현 프로그램을 유도합니다.24 또한 역학은 거시적 규모의 물리적 입력과 화학 단서를 통한 나노 규모의 분자 신호를 연결하여 다중 규모의 발달 과정을 지시합니다.25 예를 들어 특정 수용체를 통한 힘 전달은 일반적인 기계적 신호가 세포에서 발현되는 인테그린 수용체의 유형이나 ECM에 존재하는 리간드를 기반으로 특정 세포 반응으로 전달될 수 있도록 합니다. 논리적으로 기계적 자극은 특정하지 않은 경우가 많기 때문에 항상성을 유지하기 위해 기계적 전달을 신중하게 제어해야 합니다. 또한 매트릭스는 탄성 에너지를 방출하고 시간이 지남에 따라 경화, 분해 및 매트릭스 침착을 통해 변화할 수 있기 때문에 ECM은 시간과 공간이라는 4차원에서 세포에 기계적 가변 장치를 제공합니다.

전구 세포가 태아 기관의 다양한 특수 기능으로 유도되는 기관 형성 과정에서 기계적 힘이 세포의 운명 결정을 조절합니다. 생식 밴드 확장 과정은 배아 패턴 형성 유전자 중 가장 먼저 발현되는 유전자 중 하나인 트위스트의 발현을 유도하여 위[G] 및 중장 조직의 분화를 촉진하는 기계적 힘을 생성합니다.26 자궁 내 호흡 시 기관지 상피가 늘어나면서 발생하는 인장력은 폐의 평활근 발달을 돕습니다.27 모체 혈류에 의해 생성되는 전단력은 태아의 조혈과 심장 조직의 형태 형성을 촉진하고28-30, 신장 형태 형성을 촉진하기 위해 폴리시스틴-1 및 폴리시스틴-2 단백질이 필요한 일차 섬모를 통해 발달 중인 신장의 상피에 신호를 보냅니다.31 사지 발달에서는 스트레스, 변형, 정수압 및 체액 흐름이 국소 골화 및 후속 뼈 고리 형성에 선행합니다.32 근본 분자 메커니즘은 현재 연구의 주제입니다. 이러한 사례는 장기 발달이 미세 환경의 물리적 특성에 의해 어떻게 영향을 받는지를 잘 보여줍니다. 체외 줄기세포에서 이러한 발달 과정을 요약하는 것을 목표로 하는 연구는 힘을 엄격하게 제어할 수 있는 정교한 시스템을 필요로 할 것입니다.

Complex patterning depends on cell-ECM interactions

Biochemical cues initiate morphogenesis, but the formation of cell layers that become organized into defined structures in organs requires physical traction forces [G] on the ECM, the physical properties of which provide a template for organ growth. The concerted action of biochemical signals, cell intrinsic forces, and cell-ECM interactions result in highly organized patterns of development, such as fractal patterns [G] observed in branching morphogenesis.33

In submandibular salivary gland [G] branching morphogenesis, focal adhesions [G] bound to fibronectin promote assembly of fibronectin at the branching cleft through actomyosin contractility34 (FIG. 2D). Traction forces are required for branching, which suggests that the rigidity of the matrix could alter branching by changing actomyosin contractility, but it remains to be directly determined whether matrix mechanical properties can indeed modulate branching in salivary glands.

The study of mechanobiology is complex owing to mechanical stimuli affecting multiple aspects of cell behaviour, including matrix traction forces, membrane curvature, growth factor signalling pathways and cell fate. The physical properties of ECM regulate mammary gland morphogenesis in vitro by affecting cell fate. A two-dimensional (2D) system demonstrated that ECM substrates must be soft and contain laminin to maintain the expression‎ of mammary epithelial differentiation markers, whereas stiffening of the substrate or loss of laminin resulted in reduced expression‎.35 During endothelium sprouting, increased ECM stiffness and actomyosin contractility can reduce branching as they affect membrane curvature.36 Increased actomyosin contractility in a stiffer environment maintains lower membrane curvature, which impairs cell-scale branching of the endothelial cells.37 It was also shown that matrix stiffness affects biochemical signals during angiogenesis by upregulating expression‎ of vascular endothelial growth factor receptor-2 (VEGFR2).38 Future work should examine the interaction between various effects of altered mechanics. In addition to solid-like properties such as stiffness and composition, further work is required to examine the effects of time-dependent properties of ECM mechanics on organ morphogenesis, such as stress-relaxation, degradation and plasticity. Native embryonic tissues exhibit fluid-like viscoelastic properties, which probably have a role in cell organization and ECM assembly, and thus may affect mechanosensing and biochemical pathways.

Throughout embryonic and fetal development, physical interactions within the stem cell niche play a key part in maintaining stem cell populations and ensuring they persist into adult tissues. Cell-ECM adhesion via integrins maintains stem and progenitor cell pools in germline39,40 and adult epidermal niches.41 Physical stem cell-ECM interactions also regulate the positioning of stem cells within the niche architecture and with respect to their progeny, which affects fate decisions and self-renewal in the perivascular hematopoietic stem cell niche, intestinal crypt and hair follicle. 42 Over time, the ECM helps store biological information by maintaining stem cell positioning and providing a means to transduce transient molecular signals into more permanent architectural features of the niche. Extrinsic forces that result from macro-scale movement of embryonic tissues over time are transmitted to the stem cell niche to help maintain skeletal joint progenitors, which are required for proper joint cavitation and morphogenesis.43 These observations have prompted the development of in vitro physical models of the stem cell niche to improve the maintenance and expansion of pluripotent stem cells.

복잡한 패터닝은 세포-ECM 상호 작용에 따라 달라집니다.

생화학적 단서가 형태 형성을 시작하지만, 장기에서 정의된 구조로 조직화되는 세포 층의 형성에는 ECM에 물리적 견인력[G]이 필요하며, 이러한 물리적 특성은 장기 성장의 템플릿을 제공합니다. 생화학적 신호, 세포 내재적 힘, 세포-ECM 상호작용의 복합적인 작용으로 인해 분지 형태 형성에서 관찰되는 프랙탈 패턴[G]과 같이 고도로 조직화된 발달 패턴이 형성됩니다.33
턱밑 침샘[G] 분지 형태 형성에서 피브로넥틴에 결합된 국소 접착[G]은 액토미오신 수축성을 통해 분지 틈새에서 피브로넥틴의 조립을 촉진합니다34 (그림 2D). 분지에는 견인력이 필요하며, 이는 매트릭스의 강성이 액토미오신 수축성을 변화시켜 분지를 변화시킬 수 있음을 시사하지만, 매트릭스의 기계적 특성이 실제로 침샘의 분지를 조절할 수 있는지 여부는 직접적으로 결정되어야 합니다.

기계생물학 연구는 매트릭스 견인력, 막 곡률, 성장 인자 신호 경로 및 세포 운명 등 세포 행동의 여러 측면에 영향을 미치는 기계적 자극으로 인해 복잡합니다. ECM의 물리적 특성은 세포의 운명에 영향을 미쳐 시험관 내에서 유선의 형태 형성을 조절합니다. 2차원(2D) 시스템에서는 ECM 기질이 부드럽고 라미닌을 함유해야 유선 상피 분화 마커의 발현이 유지되는 반면, 기질이 경화되거나 라미닌이 손실되면 발현이 감소하는 것으로 나타났습니다.35 내피가 발아하는 동안 ECM 강성과 액토미오신 수축성이 증가하면 막 곡률에 영향을 주어 분지를 줄일 수 있습니다.36 딱딱한 환경에서 액토미오신 수축성이 증가하면 막 곡률이 낮아져 내피 세포의 세포 규모 분기가 손상됩니다.37 또한 매트릭스 강성은 혈관 내피 성장 인자 수용체-2(VEGFR2)의 발현을 상향 조절하여 혈관 신생 중 생화학 신호에 영향을 미치는 것으로 나타났습니다.38 향후 연구는 변화된 역학의 다양한 효과 간의 상호 작용을 조사해야 합니다. 강성 및 구성과 같은 고체와 같은 특성 외에도 스트레스 이완, 분해 및 가소성과 같은 ECM 역학의 시간 의존적 특성이 장기 형태 형성에 미치는 영향을 조사하기 위해서는 추가 연구가 필요합니다. 원시 배아 조직은 유체와 같은 점탄성 특성을 나타내며, 이는 아마도 세포 조직과 ECM 조립에 중요한 역할을 하여 기계감지 및 생화학적 경로에 영향을 미칠 수 있습니다.
배아 및 태아 발달 과정에서 줄기세포 틈새 내의 물리적 상호 작용은 줄기세포 집단을 유지하고 성체 조직으로 지속되도록 하는 데 중요한 역할을 합니다. 인테그린을 통한 세포-ECM 접착은 생식세포39,40 및 성체 표피 틈새에서 줄기세포와 전구세포 풀을 유지합니다.41 또한 물리적 줄기세포-ECM 상호작용은 틈새 구조 내에서 그리고 자손과 관련하여 줄기세포의 위치를 조절하여 혈관 주변 조혈 줄기세포 틈새, 장 선와 및 모낭의 운명 결정 및 자기 재생에 영향을 미칩니다. 42 시간이 지남에 따라 ECM은 줄기세포의 위치를 유지하고 일시적인 분자 신호를 틈새의 보다 영구적인 구조적 특징으로 변환하는 수단을 제공함으로써 생물학적 정보를 저장하는 데 도움을 줍니다. 시간이 지남에 따라 배아 조직의 거시적 움직임으로 인해 발생하는 외적 힘은 줄기세포 틈새로 전달되어 적절한 관절 공동화 및 형태 형성에 필요한 골격 관절 전구세포를 유지하는 데 도움이 됩니다.43 이러한 관찰은 만능 줄기세포의 유지 및 확장을 개선하기 위해 줄기세포 틈새의 체외 물리적 모델 개발을 촉진했습니다.

3. Manipulating mechanobiology

The study of embryonic and fetal development is complicated by the diverse ways in which physical forces and interactions affect stem cells. Engineering systems that act as an interface between materials and stem cells, in vitro, enable the manipulation of physical, chemical and biological parameters of the interaction. Synthetic versions of the stem cell niche have been developed to precisely investigate how mechanical forces regulate stem cell behaviour. Here we introduce the criteria for tuning mechanical stem cell-niche interactions in materials-based systems. We focus on techniques used in studies on mechanoregulation of stem cells and describe how these systems have been exploited to unravel biological mechanisms.

Challenges and criteria for building synthetic niches with tunable mechanics

Various synthetic niches are utilized to study how mechanical cues regulate stem cells in vitro as their properties can be manipulated in a more predictable manner than the niche in vivo.22,44–48 These systems can be used to study how externally applied forces impact individual cells or multi-cellular tissue models. They also enable studies of how cell intrinsic forces regulate cell behaviour, often as a feedback from the resistance to these forces provided by the niche. However, the interpretation of results obtained with these systems is challenging, and it is crucial to achieve independent control of the various physical and chemical properties of the synthetic niche.

Polystyrene is a classic cell culture model, but it is a very rigid plastic surface that adsorbs serum and cell-secreted proteins in a non-specific manner, and thus cannot be used to control physical or biochemical cues. Purified ECM proteins, such as collagen,49 laminin33 and recombinant basement membrane (Matrigel)38 are used as synthetic niches to provide a more defined and physiologic microenvironment for in vitro studies. Alternatively, decellularization of tissues yields native extracellular matrices that may have more representative physicochemical properties of the native niche.50,51 However, decellularized tissues and native ECM often have poorly defined composition, and may contain many different biochemical and physical cues, making it difficult to test specific hypotheses. Raising the concentration of ECM proteins in hydrogels is often used to increase the stiffness to a limited range, but it also increases the density of adhesion ligands available for cellular receptors. (FIG. 3A)

3. 기계생물학 조작
배아 및 태아 발달 연구는 물리적 힘과 상호 작용이 줄기세포에 영향을 미치는 다양한 방식으로 인해 복잡합니다. 체외에서 물질과 줄기세포 사이의 인터페이스 역할을 하는 엔지니어링 시스템을 통해 상호작용의 물리적, 화학적, 생물학적 매개변수를 조작할 수 있습니다. 기계적 힘이 줄기세포의 행동을 어떻게 조절하는지를 정밀하게 조사하기 위해 합성 버전의 줄기세포 틈새가 개발되었습니다. 여기에서는 재료 기반 시스템에서 기계적 줄기세포와 틈새 상호작용을 조정하기 위한 기준을 소개합니다. 줄기세포의 기계적 조절 연구에 사용되는 기술에 초점을 맞추고 이러한 시스템이 생물학적 메커니즘을 밝히는 데 어떻게 활용되었는지 설명합니다.

튜닝 가능한 메커니즘으로 합성 틈새를 구축하기 위한 도전 과제와 기준
다양한 합성 틈새는 생체 내 틈새보다 더 예측 가능한 방식으로 특성을 조작할 수 있기 때문에 기계적 단서가 시험관 내에서 줄기세포를 조절하는 방법을 연구하는 데 활용됩니다.22,44-48 이러한 시스템은 외부에서 가해지는 힘이 개별 세포 또는 다세포 조직 모델에 미치는 영향을 연구하는 데 사용할 수 있습니다. 또한 틈새가 제공하는 이러한 힘에 대한 저항의 피드백으로 세포 내재적 힘이 세포 행동을 어떻게 조절하는지에 대한 연구도 가능합니다. 그러나 이러한 시스템으로 얻은 결과를 해석하는 것은 어렵고, 합성 틈새의 다양한 물리적 및 화학적 특성을 독립적으로 제어하는 것이 중요합니다.

폴리스티렌은 고전적인 세포 배양 모델이지만 매우 단단한 플라스틱 표면으로 혈청과 세포 분비 단백질을 비특이적인 방식으로 흡착하므로 물리적 또는 생화학적 단서를 제어하는 데 사용할 수 없습니다. 콜라겐49 라미닌33 및 재조합 기저막(마트리겔)38과 같은 정제된 ECM 단백질은 체외 연구를 위한 보다 명확하고 생리학적인 미세 환경을 제공하기 위해 합성 틈새로 사용됩니다. 또는 조직의 탈세포화는 네이티브 틈새의 보다 대표적인 물리화학적 특성을 가질 수 있는 네이티브 세포 외 매트릭스를 생성합니다.50,51 그러나 탈세포화된 조직과 네이티브 ECM은 종종 구성이 제대로 정의되지 않고 다양한 생화학적 및 물리적 단서를 포함하고 있어 특정 가설을 테스트하기 어려울 수 있습니다. 하이드로젤에서 ECM 단백질의 농도를 높이는 것은 종종 강성을 제한된 범위로 증가시키는 데 사용되지만 세포 수용체에 사용할 수 있는 접착 리간드의 밀도도 증가시킵니다. (그림 3A)

Figure 3

Material systems to study stem cell mechanobiology. When engineering a synthetic niche, alterations in the overall polymer concentration may change the density of adhesion ligands, while changing crosslinking without altering the polymer content may vary the network mesh size (spacing between crosslinks), which can affect how molecules diffuse through the network. A) Artificial niches fabricated from naturally-derived ECM typically manipulate stiffness by altering the concentration of the matrix proteins, which increases ligand density and decreases mesh size in parallel. B) Synthetic polymer systems can offer independent control of stiffness and ligand density, by maintaining a constant polymer concentration while altering the crosslink density. However, the mesh size is altered in parallel. C) Matrices formed from alginate polymers can be crosslinked to various extents while maintaining constant ligand density and mesh size, and thus enable one to independently examine how matrix stiffness affects stem cells. (Inset) Crosslinking in this system occurs via cooperative sharing of divalent cations (red) in blocks of one type of sugar residue (G-block) on the chains, and increases in the number of crosslink sites occupied in the aligned blocks do not alter the architecture of the chains. D) Alginate polymer molecular weight (MW) can be used to control the viscoelasticity of an ionically-crosslinked alginate network. Low MW alginate (red arrow and box) forms into a network with less physical entanglement and overlap of the alginate chains. High MW alginate (purple arrow and box) has higher chain entanglement and overlap (shaded blue region), which decreases the ability of the polymer network dissipate stress. E) The low MW network (red line) is more viscous, shown by its rapid relaxation of stress while a constant strain is applied. The high MW network (purple line) dissipates stress more slowly due to more physical entanglement and overlap. The covalently-crosslinked network (black line) is more elastic than the viscoelastic reversibly-crosslinked alginate, and does not significantly dissipate stress over time.

Niches fabricated using synthetic or non-mammalian ECM-derived polymers enable one to decouple cell adhesion properties from gel mechanical properties. The mechanical properties of synthetic gels can be modified by altering the density of cross-links between the polymers in the system, while independently altering the density of cell adhesion ligands (FIG. 3B), which are required for cellular mechanosensing.52,53 Many studies of 2D cell culture have been performed using polyacrylamide materials coated with ECM proteins.54 For example, poly(2-hydroxyethyl methacrylate) and polyacrylamide substrates coated with recombinant basement membrane were used to tune the elasticity of the ECM to regulate mammary gland morphogenesis in vitro.35 Covalent coupling of peptides that bind integrin or other cell adhesion receptors can also be used to independently regulate adhesion potential and mechanics and control cell-cell and cell-matrix signals. The fibronectin ligand arginine-glycine-aspartate (RGD) mimics cell-matrix contacts, whereas N-cadherin ligands mimic cell-cell junctions of mesenchymal cells.22 ECM-coated polyacrylamide materials are thus suitable for the control mechanical stiffness; however, they are limited in their ability to mimic other important aspects of in vivo stem cell niches, such as their 3D organization and their ability to be remodelled by cells.

A key goal has been the engineering of defined, synthetic niche systems with complete control of mechanical, matrix and soluble cues as an alternative to animal-derived matrices for the long-term culture of functional stem cell organoids. 55 Although in the past decade stiffness was regarded as a key metric for how the matrix resists cellular traction forces to regulate stem cell fate, these studies are typically based on an often-unstated assumption that the native niche is purely elastic. However, natural matrices, such as those comprised of self-assembled collagen,56 and tissues and organs in the body, such as brain, liver, adipose tissues, coagulated bone marrow, bone fracture hematoma, and cranial sutures,57 have viscoelastic properties over various time-scales47 (Box 2). Viscoelastic materials dissipate applied forces, which can dramatically alter how the niche responds and stores cell-generated traction forces and how external mechanical forces are conveyed to cells (Box 1). Therefore, the study of stem cell mechanobiology should consider both the elastic and viscoelastic mechanical properties of synthetic niches.

The interactions of stem cells with their in vivo niche can be more accurately mimicked by 3D than 2D systems. 3D systems differ in the way cells physically interact with their immediate environment in a geometrically-confined manner and in how molecules (e.g., growth factors) diffuse and are available to cells, which can regulate autocrine and paracrine stem cell functions.58 It is challenging to control mechanical properties and changes in diffusion separately in 3D matrices, because, in a chemically-crosslinked network such as poly(ethylene glycol) (PEG) (Box 2), altering the density of cross-links also alters the material’s mesh size (FIG. 3A and B), thus affecting the diffusion of macromolecules and complicating interpretation of studies. Hydrogels fabricated from alginate enable one to decouple changes in crosslink density, and resulting stiffness, from mesh size, ligand density and diffusion properties.59 Alginate is a polysaccharide that is crosslinked by cooperative binding of divalent cations by blocks of sugar residues in adjacent polymer chains, leading to no change in the arrangement of the polymer chains as crosslinking is increased (FIG. 3C).60 Moreover, the viscoelasticity of alginate systems can be tuned by the polymer molecular weight, independent of ligand density, stiffness, degradability and transport (FIG. 3D–E).47,61–63 Thus, alginate systems are suitable to analyse how these physical parameters influence stem cell mechanobiology, whereas many other systems have limitations as these parameters cannot be regulated independently.

Lastly, it is important to recognize that mechanotransduction is not a one-way path, where mechanical cues impact cell behaviour, as ECM remodelling through the degradation and synthesis of matrix components has important roles in mechanotransduction45 (FIG. 4A). In response to increased stiffness or loading, cells can secrete matrix components or proteases that enhance or diminish adhesive interactions, stiffen or soften the ECM and activate or inactivate downstream signalling pathways.64 Synthetic and non-mammalian derived ECM materials can be engineered to either resist degradation or to be degraded in a controlled manner, by building in proteolytically labile crosslinks45,65 and/or using hydrolytically labile gels,66 in which the impact of degradation can be probed. Moreover, stem cells can remodel the surrounding artificial matrix by applying traction forces and protruding actin structures that plastically deform a viscoelastic matrix, independently of degradation (FIG. 4B–C),67 as it occurs during organ morphogenesis.

Figure 4

Three-dimensional synthetic niches physically confine stem cells and present mechanical cues that impact cell behavior and fate through forces. A) The synthetic extracellular matrix (ECM) provides resistance to cell-generated forces, and in response, stem cells can actively remodel the niche by traction-mediated deformations, degradation of cleavable domains by proteases (purple segments), and production of additional ECM. The stiffness and other mechanical cues from the synthetic matrix network can trigger a self-renewal program (blue arrow), or programs defining distinct lineages of daughter cells (red and green arrows). B) Viscoelastic synthetic niche allows cells to remodel their surrounding matrix network (red), by applying traction forces (black arrows) on matrix ligands (green circles) that allow the cell to spread and change shape by plastically, or permanently, deforming the polymer chains. C) Conversely, a purely elastic non-degradable synthetic niche (black) does not permit cells to plastically deform the polymer network and prevents cell spreading.

Several key criteria should be taken into account when engineering synthetic niches for modelling and manipulating the mechanical microenvironment of stem cells in order for these systems to provide meaningful data. Components of the synthetic niche should interact with specific cell receptors to act on well-defined mechanotransduction pathways. At the same time, it should be possible to independently control mechanical properties (such as stiffness, viscoelasticity, degradation, transport and swelling) to study their effects. Not discussed in this Review, the architecture of the ECM and topology of a material system may also impact how stem cells respond to mechanical cues.68,69

Applying extrinsic forces to directly probe mechanoresponses

Extrinsic forces can be applied to stem cells in bulk matrices that contain large numbers of cells, or at the microscale on individual or small numbers of cells. The forces can be applied in a dynamic or static manner. In many in vivo tissues, cells are subject to mechanical strain cyclically, owing to biological rhythms such as breathing, movement and blood flow, which provides mechanical cues that regulate development. Static forces often result in loading of the axial skeleton or isometric muscle contraction [G]. In synthetic systems, it is easier to apply cyclic or static strain to cells on 2D substrates.70 Compressive or tensile loads can also be applied to bulk 3D material systems using mechanical loading devices or hydrostatic pressure,71 but loading can induce convective flow [G] in the system, potentially altering nutrient availability and thus affecting cell viability or function. External loads can also be applied by acoustic waves, mechanical vibrations and microbubble cavitation, among others (Box S1).72–74

Elucidation of cellular mechanotransduction pathways may require precise control of extrinsic mechanical cues at the interface with individual or small collections of cells, which can be achieved using micro-scale probes such as micropipette aspiration,75 or magnetic devices such as twisting cytometry (Box S1).76 Magnetic probes can generate localized and repeatable extrinsic forces on the cortex of individual cells in a high-throughput and scalable manner.77 Microfluidics [G] can be used to deform single cells by extensional flow to measure their mechanical properties in a cytometer.78 Micro-scale technologies can also apply cyclic stretches on cells to mimic biological rhythms such as movement, breathing and blood flow. For example, a micro-fabricated device has been developed to mimic lung alveolar form and function by culturing a layer of epithelial cells in contact with air on one side of a porous elastic material and fluid on the other and applying controlled cyclic strain on the cells. This system can model lung disease and drug toxicity.79

External forces can also be combined with micro-scale measurement and/or control of cell-generated forces. For example, polymer micropillars on a planar substrate80 can be used to measure cytoskeletal tension and focal adhesion dynamics under equiaxial static stretch.81 Mechanical tension in cells can be stimulated by photo-actuation with thermally-responsive micropillars.82 Furthermore, micro-scale technologies can tune spatial cues to show how cell shape and spreading area is linked to how they respond to mechanics.83,84 These technologies offer the possibility to control multiple modes of external mechanical loading on stem cells. Further understanding of stem cell behaviour could be achieved by combining these technologies with sophisticated biological read-outs (e.g., single-cell and next generation sequencing, highly multiplexed mass spectrometry), advances in synthetic biology and gene editing with CRISPR to precisely control gene expression‎ in mammalian cells, as well as advances in soft robotic systems that closely mimic the physical properties of the native ECM.85

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4. Stem cells respond to forces

Stem cells respond to mechanical cues and properties of their physical niche through mechanosensing and mechanotransduction, which affects proliferation, self-renewal and differentiation into specific cell fates, as well as their self-assembly and organization (Table S2).

Principles of mechanosensing and mechanotransduction in stem cells

Stem cells sense their mechanical environment through cell-cell and cell-ECM adhesion, mechanosensitive ion channels and their primary cilium (FIG. 1A).81,86,87 Several classical pathways transduce physical cues to biochemical signals, although it is important to note that mechanotransduction can function differently depending on context and cell type. Integrin receptors bind ECM ligands such as RGD, which activates focal adhesion kinase (FAK, FIG. 1B), an important regulator of cell adhesion. In response to mechanical perturbations, mechanical homeostasis in stem cells is maintained by modifying focal adhesion ligand affinity, by regulating focal adhesion assembly and disassembly, as well as by regulating the underlying cytoskeleton and actomyosin contractility. 81 These adhesion complexes and interacting cytoskeleton activate mechano-responsive signaling, such as Rho kinase (RhoA) and mitogen-association protein kinase (MAPK), and downstream mechano-transduction pathways, such as MAL, a G-actin-binding coactivator of serum response factor (SRF),88,89 Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ)90 (FIG. 1B). Moreover, the cytoskeleton is associated with nuclear structures, linking the physical properties of cells to gene expression‎. Cytoskeletal forces are transmitted to the nucleus by the lamin A component of the nuclear lamina (LMNA, FIG. 1B), altering chromatin structure, for example inducing stretching and opening, which regulates ttranscription factor accessibility.76 LMNA expression‎ and assembly of the nuclear lamina increases with tissue stiffness.75 Extrinsic forces may also directly upregulate transcription by deforming the nucleus.91 Neural stem cells respond to cell membrane tension through a mechanically-gated Piezo1 calcium ion channel (Piezo1).86 Moreover, primary cilia have important roles in mechanoregulation in adult tissues by engaging the cytoskeleton to activate signalling pathways that promote the differentiation of stem cells in response to mechanical cues.87,92

4. 줄기세포는 힘에 반응합니다.

줄기세포는

증식, 자기 재생 및 특정 세포로의 분화,

자기 조립 및 조직에 영향을 미치는 기계 감지 및 기계 전달을 통해

물리적 틈새의 기계적 신호와 특성에 반응합니다(표 S2).

줄기세포의 기계 감지 및 기계 전달의 원리
줄기세포는 세포-세포 및 세포-ECM 접착, 기계감응성 이온 채널 및 주요 섬모를 통해 기계적 환경을 감지합니다(그림 1A).81,86,87 몇 가지 고전적인 경로가 물리적 신호를 생화학적 신호로 변환하지만, 맥락과 세포 유형에 따라 기계전달이 다르게 기능할 수 있다는 점에 유의해야 합니다. 인테그린 수용체는 세포 접착의 중요한 조절자인 국소 접착 키나아제(FAK, 그림 1B)를 활성화하는 RGD와 같은 ECM 리간드와 결합합니다. 기계적 교란에 대응하여 줄기세포의 기계적 항상성은 국소 접착 리간드 친화성을 수정하고, 국소 접착 조립 및 분해를 조절하고, 기본 세포 골격과 액토미오신 수축성을 조절함으로써 유지됩니다. 81 이러한 접착 복합체와 상호 작용하는 세포골격은 Rho 키나아제(RhoA) 및 미토겐-결합 단백질 키나아제(MAPK)와 같은 기계 반응성 신호와 혈청 반응 인자(SRF)의 G-액틴 결합 보조 인자인 MAL,88,89 예 관련 단백질(YAP) 및 PDZ 결합 모티브의 전사 보조 인자(TAZ)90 (그림 1B) 같은 다운스트림 기계 신호 전달 경로를 활성화합니다. 또한 세포 골격은 핵 구조와 연관되어 세포의 물리적 특성과 유전자 발현을 연결합니다. 세포 골격의 힘은 핵층(LMNA, 그림 1B)의 라민 A 성분에 의해 핵으로 전달되어 염색질 구조를 변화시키고, 예를 들어 신축과 개방을 유도하여 전사인자 접근성을 조절합니다.76 핵층의 LMNA 발현과 조립은 조직의 강성에 따라 증가합니다.75 외력은 핵을 변형시켜 전사를 직접 상향조절할 수도 있습니다.91 신경줄기세포는 기계적으로 게이트되는 피에조1 칼슘 이온 채널(Piezo1)을 통해 세포막 장력에 반응합니다.86 또한 원발 섬모는 세포골격과 결합하여 기계적 신호에 반응하여 줄기세포의 분화를 촉진하는 신호 경로를 활성화함으로써 성체 조직의 기계 조절에 중요한 역할을 합니다.87,92

Mechanoregulation of self-renewal and proliferation

The mechanical properties of cell adhesion substrates regulate stem cell self-renewal in culture, which is consistent with observations in developmental studies. While developmental studies have primarily examined the role of adhesion receptors, synthetic niche systems are also used to examine how the mechanical properties of the environment regulate stem cell behaviour. For example, increased stiffness of tropoelastin substrates enhanced hematopoietic stem and progenitor cell expansion, which, interestingly, was dependent on the structure and contractility of the cytoskeleton but was independent of integrin signaling.93 Similarly, the stiffness of electrospun substrates controlled iPSC proliferation.94 In adult and neonatal cardiomyocytes, compliant elastic matrices enhanced clonal expansion by suppressing maturation and promoting de-differentiation, modifying the sarcomere network, and promoting cell division.95

Synthetic niche systems have been used to examine how ECM proteins in fibrillar structures, as opposed to homogeneous and nonfibrillar matrices, can differentially regulate stem cells. Decreasing fibrillar stiffness enhanced stem cell proliferation by enhancing traction forces and ligand clustering, independent from the overall substrate mechanical properties.96 Extrinsic forces also regulate stem cell proliferation and self-renewal. Mechanical strain applied to ES cells on 2D elastomeric substrates coated with recombinant basement membrane increased their self-renewal and reduced differentiation.97 However, these 2D system do not mimic the environment of the developing embryo.

Bone marrow-derived mesenchymal stem cells (MSCs) are a useful model to study stem cell mechanobiology, as they are post-natal cells that can be isolated from adult tissues. MSCs can differentiate into multiple lineages, giving rise to tissues with a wide range of mechanical properties, including rigid bone and cartilage, and soft fat and marrow stroma.54 Uniaxial stretch of MSCs upregulated the Wnt/β-Catenin pathway, suggesting that mechanical strain promotes proliferation.98 However, further work is required to determine the magnitude and mode of strain that supports MSC proliferation. Exploiting mechanical cues to maintain and expand stem cell populations in culture is potentially important for scaling up the production of cells for therapeutic applications such as transplantation.

자가 재생 및 증식의 기계적 조절

세포 접착 기질의 기계적 특성은 배양에서 줄기세포의 자가 재생을 조절하며, 이는 발달 연구에서 관찰된 결과와 일치합니다. 발달 연구에서는 주로 접착 수용체의 역할을 조사했지만, 합성 틈새 시스템은 환경의 기계적 특성이 줄기세포의 행동을 조절하는 방법을 조사하는 데도 사용됩니다. 예를 들어, 트로포엘라스틴 기질의 강성이 증가하면 조혈 줄기세포와 전구 세포의 확장이 강화되는데, 흥미롭게도 이는 세포 골격의 구조와 수축성에 의존하지만 인테그린 신호와는 무관한 것으로 나타났습니다.93 마찬가지로, 전기방사 기질의 강성은 iPSC 증식을 제어했습니다.94 성인 및 신생아 심근세포에서 순응성 탄성 매트릭스는 성숙을 억제하고 탈분화를 촉진하며 육종 네트워크를 수정하고 세포 분열을 촉진하여 클론 확장을 향상시켰습니다.95

합성 틈새 시스템을 사용하여 동질 및 비섬유질 매트릭스와 달리 섬유소 구조의 ECM 단백질이 어떻게 줄기세포를 차별적으로 조절할 수 있는지 조사했습니다. 피브릴 강성을 감소시키면 전체 기질의 기계적 특성과는 무관하게 견인력과 리간드 클러스터링을 강화하여 줄기세포 증식을 향상시켰습니다.96 외적 힘도 줄기세포의 증식과 자기 재생을 조절합니다. 재조합 기저막으로 코팅된 2D 엘라스토머 기판에서 ES 세포에 기계적 변형을 가하면 자기 재생이 증가하고 분화가 감소했습니다.97 그러나 이러한 2D 시스템은 발달 중인 배아의 환경을 모방하지 않습니다.

골수 유래 중간엽 줄기세포(MSC)는 성인 조직에서 분리할 수 있는 출생 후 세포이기 때문에 줄기세포 기계생물학을 연구하는 데 유용한 모델입니다. MSC는 여러 계통으로 분화하여 단단한 뼈와 연골, 부드러운 지방과 골수 기질 등 다양한 기계적 특성을 가진 조직을 생성할 수 있습니다.54 MSC의 일축 스트레치는 Wnt/β-Catenin 경로를 상향 조절하여 기계적 변형이 증식을 촉진한다는 것을 시사합니다.98 그러나 MSC 증식을 지원하는 변형의 크기와 방식을 결정하려면 추가 연구가 필요합니다. 배양 중인 줄기세포 집단을 유지하고 확장하기 위해 기계적 단서를 활용하는 것은 이식과 같은 치료용 세포의 생산을 확대하는 데 잠재적으로 중요합니다.

Mechanoregulation of stem cell fate in synthetic niches

Intrinsic mechanical signals play an important part in the generation of microtissues from ES and iPS. The differentiation state of microtissues derived from ES cells increases with the stiffness and viscosity of the cell aggregate, which is regulated by actomyosin contractility.99 Soft collagen-coated polyacrylamide 2D substrates that matched the intrinsic softness of ES cells maintained their pluripotency,100 whereas localized stresses externally applied to the surface of ES cells by RGD ligand-coated magnetic beads induced ES cell spreading and differentiation.101 The responses were correlated with the intrinsic softness of the cell,101 probably by regulating actomyosin contractility.

The spatial orientation of ES cell microtissues guides the formation of embryonic germ layers, which is mediated by cell-cell contact and cortex contractility that transmits forces in the cell layers.102 3D scaffolds that encapsulate ES cell-derived embryoid structures tune cell fate and germ layer specification by enabling the manipulation of cell-generated forces.103 By mimicking the soft biophysical architecture of embryoid bodies early in development, artificial niches were used to guide iPSC self-organization to promote amniogenesis in a BMP-SMAD-dependent manner.104 Moreover, a soft micropillar substrate system was used to regulate the Hippo pathway in iPS cells to promote neural induction, similar to what is observed in embryonic development during neural plate specification and A-P axis formation during neurulation.105

Defined mechanical and biochemical cues of synthetic niches can also facilitate the reprogramming of somatic cells into iPS cells, as indicated by the ability of soft ECM with a particular composition to regulate mesenchymal-to-epithelial transition and epigenetic remodeling to enhance reprogramming.106 It remains to be determined specifically how viscoelastic properties regulate in vitro generation of microtissues from stem cells, although these are likely to play a prominent role. Recent work showed expansion of stem cells and development of structures in a synthetic PEG system with reversible crosslinks that are expected to impart viscoelasticity, although the mechanics were not yet characterized.107

Mechanical cues regulate the fate of stem and progenitor cells isolated from adult tissues

Many studies demonstrate that the differentiation of MSCs derived from adipose or bone marrow tissue is regulated by the elasticity of their matrix,44,54 and MSCs appear to switch to the fate of cells whose native ECM is most closely matched to the elasticity of the substrate or matrix, i.e., stiffer substrates promote osteogenic differentiation, while softer substrates induce fat or neuronal differentiation.54 Hydrogel macroscale mechanics directly contribute to differentiation fate, which is jointly regulated by the specific ECM proteins or immobilized ligands utilized for adhesion.52 N-cadherin ligands that mimic cell-cell adhesive junctions of MSCs increased the mechanical threshold for YAP/TAZ signaling (FIG. 1B) and reduced actomyosin contractility compared to only RGD ligands that mimic cell-matrix adhesion.22 In other cell types, substrates that match the soft mechanics of bone marrow promoted the differentiation of megakaryocytes and generation of proplatelets.108 Neural stem cells were directed towards neuronal differentiation in soft conditions, and glial cells in stiffer conditions.109 These findings have been extended to 3D systems, and the use of alginate-based systems demonstrated that MSC fate in 3D is controlled independently by stiffness, while diffusion and overall gel architecture were held constant.44 The development of artificial 3D ECMs has also revealed that mechanical and matrix factors contribute to the programming of adult stem cells in organoids.55 Highly stiff synthetic hydrogels enhanced the expansion of adult intestinal stem cells in a YAP-dependent manner, while low matrix stiffness promoted cell differentiation and intestinal organoid formation. The spatial properties and heterogeneities of the stem cell niche affect how stem cells transduce mechanical cues into changes of cell fate, as more random, less-ordered mechanical cues suppressed differentiation markers of MSCs and maintained stem cell lineage markers compared to a more ordered structure.110 Furthermore, MSCs may be more responsive to mechanical gradients of stiffness, which can be established by varying the level of crosslinking along a spatial axis, as compared to stiffness alone.111–113

The impact of mechanics on cell fate appears to be regulated in a cell-intrinsic manner by YAP/TAZ signaling, which allows cells to store information from the past physical environments to influence cell fate and suggests they possess a mechanical memory.114 Retention of mechanical history in MSCs is regulated by mi-R21 levels (FIG. 1B), which gradually increased during priming on stiff substrates to promote their fibrogenic program.115 MSCs may also integrate the mechanical properties over time, as earlier stiffening induced more osteogenic differentiation compared to later stiffening in a dynamic system.116

In addition to stiffness, or elasticity, the ability of the ECM to flow and dissipate stress, or viscoelasticity (Box 1), can regulate the spreading behaviour and differentiation of MSCs through traction forces in a cell intrinsic manner.47,117 Matrices that rapidly relax an applied stress can enhance osteogenic differentiation and matrix production by MSCs. Additionally, substrate creep [G] and stress-stiffening [G] (Box 2) affect MSCs differentiation.48,118 Unlike purely elastic systems, physical environments that exhibit viscoelastic behaviour allow cells to reversibly change their shape and volume in response to cues (FIG. 4B–C). Changes in cell volume may be partially responsible for the impact of mechanics on stem cell fate. These findings are consistent with the role of mechanics in regulating signaling over space and time, and cells’ dependence on re-organization and/or dissipation of energy in the matrix.

Externally applied forces also can regulate the fate of adult stem cells, although the precise mechanotransduction pathways are often unclear. Articular and vertebral cartilage are subject to loading from movement and forces transmitted through the axial skeleton, and synthetic niche systems are used to investigate how external forces, like hydrostatic pressure, promote chondrogenesis and matrix production of stem cells in vitro. 119 Mechanoregulation of chondrogenesis also appears to be coupled to the rate- and time-dependence of loading. Dynamic compression of MSCs by cyclic compressive strain (10%, 1 Hz) increased the activity of downstream TGF-β1 signaling involving the Smad-2/3 pathways, and promoted chondrogenesis.120 The axial skeleton is also subject to external loading, and mechanical stimulation by cyclic stretching, which induces Notch signaling, has been found to promote osteogenic differentiation of MSCs and to suppress histone deacetylase expression‎, providing an additional potential mechanism for regulation of mechanotransduction in MSCs.121 Furthermore, fluid shear stress enhanced the osteogenic potential of MSCs.122,123 However, it is important to note that hydrogels may dissipate stress, so less force may be transmitted to encapsulated cells than believed, and cytoskeletal changes in response to external stimuli may change how mechanical input is perceived or felt by the cell. Further studies are required to determine how these external cues are sensed by stem cells and transduced into mechanoresponses. These challenges could possibly be addressed by integrating traction force microscopy with macroscale mechanical stimuli, allowing one to observe cytoskeletal dynamics in response to external loading, or the use of mechanically-sensitive molecular probes to measure ligand-receptor interactions in response to macroscale loading.

5. Regenerative medicine applications

Regenerative medicine aims to treat injuries or disease with therapies that repair or replace organ or tissue function. Many approaches focus on the use of endogenous or transplanted stem cells, and controlling cell fate stability is likely key to successful regeneration. Following systemic or local transplantation, stem cells often lose viability or regenerative potential, or are cleared in the lungs, liver and spleen. Safety concerns are paramount with the use of pluripotent cells, highlighting the importance of controlling their localization and fate in vivo.

Exploiting cell intrinsic forces to enhance tissue regeneration

Synthetic matrices with defined mechanical properties and biophysical properties are used to prime stem cells ex vivo prior to transplantation to improve their capacity for regeneration (FIG. 5A), as well as control their behavior in vivo following transplantation (FIG. 5B). Priming hematopoietic stem and progenitor cells with materials of specific mechanical properties can increase their yield following transplantation and enhance reconstitution.93,124 Hydrogel substrates (2D PEG substrate coated with laminin) with a stiffness value that mimicked that of muscle promoted muscle stem cell self-renewal and increased their regenerative capacity when transplanted. 125 Substrate stiffness may also affect how stem cells regulate immune responses, for example by modifying the secretory profile of MSCs. 126 Soft elastic materials in 2D, composed of fibronectin-coated polyacrylamide, induced reactive oxygen species (ROS) signaling in human bone marrow MSCs, whose conditioned media was shown to improve wound healing in vivo. 127 In vivo, soft alginate-based scaffolds with matrix metalloprotease (MMP)-cleavable domains improved MSC invasion into host tissue in mouse models compared to a stiffer material, and matrix production at the delivery site was significantly enhanced. 65 In engineering 3D human cardiac tissues, enhanced sarcomere [G] organization and contractile function resulted from the use of a chemically-crosslinked gelatin-PEG material with stiffness similar to native cardiac tissue that facilitated the ability of cells to establish cell-cell adhesions. 128 Gelatin degradation over time was likely important to these effects, because chemical crosslinking of PEG and gelatin initially leads to a purely elastic material that would not otherwise allow cells to self-organize or remodel matrix. Synthetic niches can improve MSC transplantation for bone tissue repair by tuning stiffness to direct cell fate, as well as programming porosity to facilitate host integration, using a viscoelastic alginate system129 (FIG. 5B). Microfluidic platforms can form synthetic niches on the single-cell level with specified stiffness to improve the distribution and paracrine function of MSCs after transplantation into the systemic circulation of animals. 130 An alginate matrix with rapid relaxation has been shown to enhance bone regeneration compared to more slowly relaxing hydrogels, even in the absence of stem cell delivery, presumably by promoting osteoblast differentiation and matrix remodeling. 131 Similarly, an appropriate combination of a material’s chemical and physical properties can regulate stem cell migration into a site of injury, and be used to support rapid cutaneous tissue regeneration. 132 The challenge of controlling stem cell fate and morphogenesis after transplantation may be addressed by harnessing the potential for organogenesis in vitro. Mechanical cues have been shown to promote organogenesis,55 so synthetic niches may be able to generate organoid tissues in vitro for subsequent repair or replacement of damaged organs in vivo (FIG. 5C).

Figure 5

Tissue regeneration can be enhanced by exploiting stem cell mechanobiology. A) Stem cells can be pre-conditioned with mechanical cues, either by culturing on or in matrices with specific stiffness or viscoelasticity, or by applying external forces of a desired strain and rate to the substrate, prior to collection of cells for localized (e.g., skeletal muscle site) or systemic delivery. B) Stem cells can instead be transplanted on or in synthetic matrices with defined mechanical properties, such as stiffness and viscoelasticity, that promote proliferation and/or a particular stem cell fate; in this example, stem cells are programmed by the matrix to enhance bone repair in the mandible. C) Mechanical cues of stiffness and viscoelasticity can also be used in vitro to mimic embryonic development by driving stem cells to undergo self-organization, differentiation, and morphogenesis into organoid tissues, which could then be subsequently transplanted for organ repair or replacement; in this example, repair of the colon. D) Direct application of externally applied forces can be utilized to enhance regeneration by endogenous stem cells; in this example, stem cells in injured skeletal muscle tissue. Both the absolute magnitude of strain and rate of application may regulate the regenerative process.

Mechanically loading cells and tissues to enhance tissue regeneration

It has long been known that external mechanical forces regulate responses to injury. Mechanical unloading of bone in microgravity favors maintenance and expansion of mesenchymal and hematopoietic stem cells in bone marrow, while limiting their differentiation,133 and disuse atrophy is observed in skeletal muscle. Current clinical practices exploit active mechanical cues on a regular basis to drive tissue remodeling and regeneration. Distraction osteogenesis, which applies external strain on segments of bone, has been widely used clinically to promote bone formation. 134 Orthopaedic implants have been designed to minimize strain-shielding at a fracture site, in order to promote strain-mediated effects on bone remodeling and regeneration. 135 A recent clinical trial showed low-magnitude, high-frequency mechanical stimulation in pediatric cancer survivors with low bone mineral density was safe and efficacious in enhancing peak bone mass during youth. 136 In dentistry, orthodontic tooth movement is achieved by stressing the periodontal ligament to induce tissue remodeling. 137

Success to date in the clinic with selected tissues generates the question of whether mechanical therapies can be extended to other disease and tissue contexts, such as muscle, and motivates the development of new therapies that exploit stem cell mechanobiology with actuating rigid or soft materials. Cyclic external strain has been used to pre-condition muscle stem cells in 3D animal-derived matrices prior to implantation, and improved the function of subsequently regenerated muscle in mouse models138,139 (FIG. 5A). However, it is not clear precisely how strain impacts cells in these systems, because the mechanics and composition of these materials are variable and not completely controlled. Alternatively, muscle can also be directly stimulated in vivo with externally applied forces to promote healing and reduce inflammatory injury (FIG. 5D). Applying forces to muscle during massage reduced inflammatory injury caused by exercise by activating FAK and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways (FIG. 1B), which increase mitochondrial biogenesis and inhibit nuclear translocation of nuclear factor κβ (p65).140 Repeated forces applied via soft robotic devices were found to promote skeletal muscle regeneration in animals following severe injury, and reduced fibrosis and inflammation.141 However, it remains to be shown clinically whether and how external stimulation impacts stem cells in muscle. An important goal of future work is to connect clinical data with pre-clinical findings relating mechanical stimuli and cell responses.

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6. Summary, Conclusions and Perspectives

Biological tissues are composed of materials, which transmit forces and exhibit diverse mechanical properties, and cells that generate and are subject to physical forces. These mechanical properties and forces regulate stem cell function and guide developmental processes, and tissue repair. As one can independently manipulate specific biophysical properties of synthetic matrices, these systems are advancing our understanding of mechanotransduction mechanisms in stem cells, and leading to technologies to guide stem cell fate. However, care must be taken when comparing results from studies as parameters such as dimensionality, chemistry, viscoelastic properties, degradability, and diffusional transport may vary substantially. Moreover, reported values of mechanical parameters can be biased by the measurement technique (Box 2). Lastly, many research groups may not have access to these synthetic systems owing to their limited commercial availability; therefore a need exists for standardized and more easily accessible model systems.

Stem cell transplantation has previously been limited by lack of control over distribution and loss of cell viability, and while biomaterials can address certain of these challenges,130 future efforts should exploit mechanics to control post-transplant cell fate. Beyond regeneration, materials systems that control the physical and biochemical microenvironment of individual cells open up exciting opportunities in stem cell research. For example, they may be valuable in building human-based in vitro models of organs and disease. New chemistries are being discovered to expand the limited repertoire of materials for stem cell culture by using screening techniques adapted from the microfabrication industry. 142 Finally, additive manufacturing can fabricate devices and material systems from computer-aided designs that mimic organ-level physiology and disease. 143

Mechanical cues likely play key roles in many other processes in biology and disease, and the advances to date in the stem cell field may find broad application in these areas as well. For example, fibrosis and cancer feature changes in the physical interactions of cells and their matrix. Fibrosis is involved in many disease processes, but it is unclear how mechanical interactions of putative cells and their niche regulate initiation and development of disease. While mechanics has been implicated in the initiation and progression of malignant lesions, 144 studies are just now demonstrating how intrinsic or extrinsic forces and the tumor microenvironment regulate tumor rejection or resistance to therapies,145 and how mechanical forces are involved in oncogenic signalling pathways. 146 Future work will bridge the gap between basic research in mechanobiology and improved therapies for patients.

6. 요약, 결론 및 전망

생체 조직은

힘을 전달하고 다양한 기계적 특성을 나타내는 물질과

물리적 힘을 생성하고 영향을 받는 세포로 구성되어 있습니다.

이러한 기계적 특성과 힘은

줄기세포의 기능을 조절하고

발달 과정과 조직 회복을 유도합니다.

Biological tissues are composed of materials, which transmit forces and exhibit diverse mechanical properties, and cells that generate and are subject to physical forces. These mechanical properties and forces regulate stem cell function and guide developmental processes, and tissue repair.

합성 매트릭스의 특정 생물물리학적 특성을 독립적으로 조작할 수 있기 때문에

이러한 시스템은 줄기세포의 기계 전달 메커니즘에 대한 이해를 발전시키고

줄기세포의 운명을 안내하는 기술로 이어지고 있습니다.

그러나

치수, 화학, 점탄성, 분해성, 확산 수송과 같은 매개변수가 크게 다를 수 있으므로

연구 결과를 비교할 때는 주의가 필요합니다.

또한 기계적 파라미터의 보고된 값은 측정 기법에 따라 편향될 수 있습니다(상자 2).

마지막으로,

많은 연구 그룹이

이러한 합성 시스템의 상업적 가용성이 제한되어 있기 때문에

이러한 합성 시스템에 접근하지 못할 수 있으므로

표준화되고 보다 쉽게 접근할 수 있는 모델 시스템이 필요합니다.

줄기세포 이식은

이전에는 세포의 분포와 생존력 손실에 대한 제어가 부족하여 제한적이었으며,

생체 재료가 이러한 문제를 일부 해결할 수 있지만,130

향후에는 이식 후 세포의 운명을 제어하기 위한

역학을 활용해야 합니다.

재생 외에도

개별 세포의 물리적, 생화학적 미세환경을 제어하는 재료 시스템은

줄기세포 연구에 흥미로운 기회를 열어줍니다.

예를 들어,

인체 기반 장기 및 질병의 체외 모델을 구축하는 데 유용할 수 있습니다.

미세 제조 산업에서 채택한 스크리닝 기술을 사용하여 줄기세포 배양용 재료의 제한된 레퍼토리를 확장하기 위한 새로운 화학 물질이 발견되고 있습니다. 142 마지막으로 적층 제조는 장기 수준의 생리와 질병을 모방한 컴퓨터 지원 설계로 장치와 재료 시스템을 제작할 수 있습니다. 143

기계적 단서는

생물학과 질병의 다른 많은 과정에서 중요한 역할을 할 수 있으며,

현재까지 줄기세포 분야의 발전은

이러한 분야에서도 광범위하게 적용될 수 있습니다.

예를 들어,

섬유증과 암은 세포와 그 기질의 물리적 상호작용에 변화를 일으킵니다.

섬유화는

많은 질병 과정에 관여하지만,

추정 세포와 그 틈새의 기계적 상호작용이

질병의 시작과 발달을 어떻게 조절하는지는 불분명합니다.

악성 병변의 시작과 진행에 역학이 관련되어 있지만, 144

내재적 또는 외재적 힘과 종양 미세 환경이 종양 거부 또는 치료에 대한 저항을 어떻게 조절하는지,145

그리고 기계적 힘이 발암성 신호 경로에 어떻게 관여하는지 보여주는 연구가

이제 막 시작되고 있습니다. 146

향후 연구는

기계생물학의 기초 연구와 환자를 위한

개선된 치료법 사이의 간극을 메울 것입니다.

Key points

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Supplementary Material

Supplementary Information

Click here to view.(48K, docx)

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Acknowledgments

We’d like to thank Dr. Jianyu Li for assistance with revising this manuscript and David Zhang for input on the figures. Funding was provided by the National Institute of Dental & Craniofacial Research of the National Institutes of Health under Award Numbers 5R01DE013033 (DM) and K08DE025292 (KV). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Glossary

cadherin-catenin complexes	Complexes of cellular receptors termed cadherins, which bind to other cells, with β-catenin, an intracellular molecule, that connect to the actin cytoskeleton in epithelial tissues to convey forces between cells.
dorsal closure	Closure of a dorsal epidermal opening that is initially formed naturally during embryonic development of Drosophila melanogaster; this process has similarity to wound healing in mammals.
cortical tension	Actomyosin-generated forces cause tension in the cytoskeleton of cells, which contributes to their shape and mechanical properties.
Rho-associated protein kinase	A serine-threonine kinase that can regulate actomyosin contractility and is downstream of RhoA and other pathways.
Stomodeum	A frontal opening in the developing embryo that forms a primordial mouth, separated from the pharynx by an oropharyngeal membrane.
traction forces	Forces on ECM or other cells generated by receptor-binding and actomyosin contractility.
fractal patterns	Highly branched geometry that is formed from repeated symmetric branching, often across multiple length scales.
submandibular salivary gland	One of the major salivary glands. Features a branched ductal structure that opens into the oral cavity, with secretory end pieces, called acini, that produce saliva by secretion of water, salts, proteins, and other macromolecules.
focal adhesions	Complexes of matrix receptors, actin cytoskeleton, and other cytoskeletal and signaling molecules that link the cytoskeleton to ECM ligands.
isometric muscle contraction	Forces generated by muscle while maintaining constant muscle length and joint angle.
convective flow	Fluid flow that transfers mass and/or heat down a fluid pressure gradient.
Microfluidics	Precise control of fluid shear forces and flow rates in micro-scale geometries, such as micro-channels.
substrate creep	Deformation, or flow, of a material during a constant application of stress.
stress-stiffening	Mechanical stiffening of a polymer network with increasing strain.
Sarcomere	Fundamental active unit in skeletal muscle that generates force from overlapping striations of actin and myosin

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Biographies

• 

Prof. Mooney is Robert P. Pinkas Family Professor of Bioengineering and Wyss Institute Core Faculty Member. His laboratory studies how environmental signals are sensed by and impact cells, in order to design and synthesize new biomaterials for a variety of applications, including mechanotransduction studies, regenerative medicine, and cancer therapies.

• 

Dr. Vining is a Bioengineering Fellow and Ph.D. candidate. His research aims to determine how the physical microenvironment of cells can regulate immune responses in regeneration and cancer. He previously completed his D.D.S. at the University Minnesota, as well as a fellowship in the NIH Medical Research Scholars Program.

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Footnotes

COMPETING INTERESTS STATEMENT

The authors declare no competing interests.

AUTHORS CONTRIBUTION:

D. J. M. and K. H. V. researched data for the article, contributed to discussion of the content, wrote the article and reviewed and/or edited the manuscript before submission.

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