This research was supported by a grant from the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), which was funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: HI16C1559).

The protocol was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Institutional Review Board at CHA Bundang Medical Center.
1. Degenerative Tendon Tissue Harvest from the Patient
<ol>
	<li>Diagnose lateral epicondylitis by taking a medical history and using physical exam findings: tenderness over the tendinous origin close to the lateral epicondyle; pain evoked by resisted extension of the wrist joint.</li>
	<li>Use the following inclusion criteria: over 18 years of age; a history of lateral epicondylitis for a minimum of six months; recalcitrant for non-operative treatment; at least three months have passed since injection of corticosteroid, platelet rich plasma, or botulinum toxin</li>
	<li>Exclude the following: pregnant women; patients with a history of cervical spine disease, elbow arthritis, rheumatologic disease, surgery around elbow, and nerve entrapment syndrome; patients refusing to donate tissue.</li>
	<li>Surgical technique 
	&#8203;NOTE: See Figure 124.
	<ol>
		<li>After general anesthesia by isoflurane and endotracheal intubation, put the arm on the table and apply a tourniquet. Then apply betadine agent on the whole arm and drape with an aseptic surgical sheet.</li>
		<li>Expose the lateral elbow through a 3&#8211;5 cm incision with No. 15 surgical scalpel blade just anteromedial to the lateral epicondyle and proximal to the level of the joint.</li>
		<li>Visually identify the extensor carpi radialis longus (ECRL) and extensor aponeurosis interface and make a splitting incision 2&#8211;3 mm in depth between the ECRL and extensor aponeurosis with a No. 15 surgical scalpel blade at the identified interface.</li>
		<li>Extract the ECRL anteriorly, which will bring the pathologic extensor carpi radialis longus brevis (ECRB) origin below into direct view.</li>
		<li>Visually identify the pathologic degenerative tissue by its characteristic dull grayish color and usually edematous and friable tissue.</li>
		<li>After identification, carefully resect all degenerative tissue sharply using No. 15 surgical scalpel and mini-blades.</li>
		<li>Embed the resected tissue (about 1 cm3) into phosphate buffered saline (PBS) immediately and remove all the surrounding tissue except tendon tissue carefully into about 10 mL of PBS as soon as possible.</li>
		<li>In cases with bony exostosis at the lateral epicondyle, remove exostosis using a rongeur and smoothen it with a rasp. Make multiple drill holes (diameter 1.6 mm, about 6&#8211;8 holes) in the anterolateral condyle to enhance vascular supply. This is optional.</li>
		<li>Close the wound layer by layer using non-absorbable suture material.</li>
	</ol>
	</li>
	<li>Healthy control tenocyte
	<ol>
		<li>Obtain healthy tenocytes from the remaining tendon of the auto-hamstring or auto-patella tendon during anterior cruciate ligament reconstruction, and use it as the control for the following experiment25. 
		NOTE: The number of samples used in this study were three in each group.</li>
	</ol>
	</li>
</ol>
2. Histology
<ol>
	<li>Store about half of the harvested tissue in neutral buffered 10% formalin in a chemical fume hood or equivalent.</li>
	<li>Embed the tissue in a paraffin block (4&#8211;5 mL) and section it in 4&#8211;5 &#181;m sections coronally.</li>
	<li>Stain sections from control and degenerative tendon with Hematoxylin and Eosin stain (H&#38;E) and Alcian blue stain at a pH of 2.5. Perform H&#38;E staining in an automated stain machine as follows.
	<ol>
		<li>Deparaffinize using xylene (3 times, 5 min, respectively).</li>
		<li>Hydrate from graded alcohols to water (100% alcohol, 4 min; 100% alcohol, 3 min; 95% alcohol, 2 min; 70% alcohol, 1 min; water, 2 min).</li>
		<li>Stain in Hematoxylin for 5 min. Wash well in water for 2 min.</li>
		<li>Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 3 s. Wash well in water for 3 min.</li>
		<li>Neutralize HCl by dipping in an 0.5% ammonia for 10 s, followed by a 3 min water wash.</li>
		<li>Stain in Eosion for 5 min.</li>
		<li>Dehydrate through alcohols (80% alcohol, 30 s; 95% alcohol, 1 min; 100% alcohol 1 min, 100% alcohol 2 min).</li>
		<li>Clear in xylene (3 times, 2 min, 2 min, and 3 min, respectively).</li>
		<li>Mount with a cover slide.</li>
	</ol>
	</li>
	<li>Perform immunohistochemitry (IHC) using the Bond Polymer Refine Detection kit according to the manufacturer&#39;s instructions with minor modifications.
	<ol>
		<li>After deparaffinization using xylene, perform an antigen retrieval procedure using Bond Epitope Retrieval Solution for 30 min at 100 &#176;C. The Bond Epitope Retrieval Solution contains a citrate based buffer and surfactant (1 L, pH 5.9&#8211;6.1).</li>
		<li>Quench endogenous peroxidases by incubating the tissue with hydrogen peroxide for 5 min.</li>
		<li>Incubate the sections for 15 min at an ambient temperature (21-22 &#176;C) with primary antibodies against vascular endothelial growth factor (VEGF) (1:200), tenomodulin (1:200), or mohawk (1:200).</li>
		<li>Perform secondary antibody tagging with a biotin-free polymeric horseradish peroxidase-linker antibody conjugate system.
		<ol>
			<li>Use secondary rabbit anti mouse IgG in 10% (v/v) animal serum in Tris-buffered saline/0.09% 2-Methyl-4-isothiazolin-3-one solution to localize mouse antibodies.</li>
			<li>Use polymer anti-rabbit Poly-HRP-IgG (&#60;25 &#181;g/mL) containing 10% (v/v) animal serum in Tris-buffered saline/0.09% to localize rabbit antibodies.</li>
			<li>Use 3,3&#39;-Diaminobenzidine tetrahydrochloride hydrate in a stabilizer solution to visualize the complex via a brown precipitate.</li>
			<li>Counterstain with &#60;0.1% hematoxylin (blue) to visualize the cell nuclei.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Observe the slides stained with H&#38;E, Alcian blue stains, and IHC for VEGF, tenomodulin, and mohawk under light microscope and obtain representative microscopic images of each slide under one high power field (400X).</li>
</ol>
3. Tenocyte Culture
<ol>
	<li>Tissue preparation
	<ol>
		<li>Prepare the tendon sample by removing all the surrounding tissue thoroughly with micro-scissors and forceps.</li>
		<li>Wash with PBS and mince into small pieces (about 1 mm3 or less).</li>
		<li>Digest tissue for 1 h in 30 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 30 mg/mL Collagenase II at 37 &#176;C.</li>
		<li>Pass cells through a 100 &#181;m cell strainer.</li>
		<li>Deactivate Collagenase II by adding 1 mL of fetal bovine serum (FBS).</li>
		<li>Centrifuge at 196 x g for 5 min.</li>
	</ol>
	</li>
	<li>Cell culture
	<ol>
		<li>Seed 3 x 105 cells into a 100 mm dish and culture with DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic solution at 37 &#176;C in a 5% CO2 incubator for 1 week.</li>
		<li>Change the culture medium every three days until 80% confluence occurs. Culture both healthy control and degenerative cell samples until passage four and use for experiment.</li>
	</ol>
	</li>
</ol>
4. Immunocytochemistry (ICC)
<ol>
	<li>Transfer 200 &#181;L of cultured cell (2 x 105 cells) to the eight wells of a chamber slide and grow the cells to confluence with the addition of fresh media for 1 day.</li>
	<li>Wash the cells with PBS and fix with 500 &#181;L of 4% formaldehyde per well.</li>
	<li>Incubate the slides with 0.5% Triton X-100 in PBS for 10 min for permeabilization of the membranes.</li>
	<li>Block in PBS with 1% bovine serum albumin and 0.05% TWEEN-20 for mohawk and tenomodulin, respectively.</li>
	<li>Incubate in the dark with mouse anti-mohawk monoclonal antibody (1:100 dilution) and goat anti-tenomodulin antibody (1:100 dilution) at 4 &#176;C overnight.</li>
	<li>Incubate the secondary antibodies (Alexa 555-anti goat (1:200) for tenomodulin; Alexa 488-anti mouse (1:200) for mohawk) in the dark for 1 h at room temperature.</li>
	<li>Mount with 0.5 &#181;L of a fluorescence mounting medium.</li>
	<li>Analyze using a fluorescence microscope (400X).</li>
</ol>
5. Proliferation Assay
<ol>
	<li>Measure cell viabilities using water-soluble tetrazolium (WST)-1.
	<ol>
		<li>Seed both healthy and degenerative cells into 96 well plates at a density of 2 x 103 cells per well for 24, 72, and 120 h.</li>
		<li>After incubation, add 10 &#181;L of the 10% WST-1 solution to each well.</li>
		<li>Incubate the plates for 3 h at 37 &#176;C in 5% CO2.</li>
		<li>Measure the optical densities at 450 nm using a microplate reader. Take readings at least three times at all time points for each sample.</li>
	</ol>
	</li>
	<li>Measure cellular DNA amount using the BrdU assay.
	<ol>
		<li>Seed both healthy and degenerative cell into 96 well plates at a density of 2 x 103 cells per well for 24, 72, and 120 h.</li>
		<li>After incubation, add 10 &#181;L of 10% BrdU solution to each well.</li>
		<li>Incubate the plates for 3 h at 37 &#176;C in 5% CO2.</li>
		<li>Add 100 &#181;L/well of the Fixing/Denaturing Solution (60&#8211;70% ethanol, 0.1&#8211;0.5% sodium hydroxide) and keep the plate at room temperature for 30 min.</li>
		<li>Add 100 &#181;L/well prepared 1x detection antibody solution and keep the plate at room temperature for 1 h.</li>
		<li>Remove solution and wash plate three times with 1x Wash Buffer.</li>
		<li>Add 100 &#181;L/well prepared 1x HRP-conjugated secondary antibody solution and keep the plate at room temperature for 30 min.</li>
		<li>Remove the solution and wash plate three times with 1x Wash Buffer.</li>
		<li>Add 100 &#181;L of 50% TMB Substrate.</li>
		<li>Incubate for 30 min at room temperature.</li>
		<li>Add 100 &#181;L of STOP Solution.</li>
		<li>Measure the optical densities at 450 nm using a microplate reader. 
		NOTE: Take readings at least three times at all time points for each sample.</li>
	</ol>
	</li>
</ol>
6. Statistical Analysis
<ol>
	<li>Collect and analyze data using SPSS.</li>
	<li>Depict data by mean &#177; standard error of mean.</li>
	<li>Use Mann Whitney U test.</li>
	<li>Consider differences statistically significant when the p value is less than 0.05 (p &#60;0.05).</li>
</ol>

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The authors have nothing to disclose.

A number of previous studies have reported how to create chronic tendinopathic animal models using different procedures such as collagenase or kartogenin injection, treadmill running, and more26,27. Although numerous studies showed promising therapeutic effects based on these animal models, experiments using the human degenerative tenocyte would be crucial in the field of tendinopathy in order to reproduce the efficacy of treatment. In this article, we establishe...

Tendinopathy is a chronic degenerative musculoskeletal condition that develops in various parts of the body. Recently, the number of cases of tendinopathy has increased greatly in the developed world due to growing participation in recreational sports and increased life expectancy1,2. The cause of tendinopathy is considered to be multifactorial and these causes include ischemia, oxygen free radical injuries, an imbalance between vasoconstrictor and vasodilator innervations, internal micro-tears, and changes to neuro-regulation3,4,5,6,7,8. Most treatments for tendinopathy only relieve its symptoms. Moreover, treatments without tissue regeneration require a long time for rehabilitation and achieve a limited response from injured tendons, which imposes a clinical challenge for physicians9.
The incompetence of current treatment options along with the lack of the degenerative tendon&#39;s ability to self-heal has lead researchers to take interest in exploring alternative treatment strategies. Recently, new studies reported many promising results for enhancing the healing efficacy of the tendinopathy tendons using growth factors, stem cell based therapy, and gene therapy10,11,12.
Through a literature review, we found that the involved studies may be divided into two categories based on their analysis materials: animal models such as a rat, a mouse, or a rabbit; and human models. With regard to the animal model, currently there are two popular techniques to generate tendinopathy: chemical induction of injury or mechanical overloading the model. However, each animal model was limited in reproducing the complex human tendinopathy pathology13,14.
Most papers using human samples were analyzed histologically or performed the in vitro experiment based on a healthy human tenocyte instead of a degenerative tenocyte15,16,17,18,19,20,21. Only a few papers reported that they used a human degenerative tenocyte, but they did not describe in detail the protocol used to get the degenerative tenocyte from the human22,23. In this context, it should be noted that successful results from either the animal model or the healthy tissue/tenocyte may not necessarily predict human efficacy or efficacious dosing because tendon degeneration is a complicated process and the pathogenesis is still not fully understood.
Collectively, it is necessary to describe the standard protocol for obtaining the degenerative tenocyte from human tissue without causing adverse effects to the donor. This article describes a protocol on how to acquire the human degenerative tenocyte. To validate the protocol, the harvested tissues were analyzed histologically. Then, the cultured cell was confirmed as the degenerative tenocyte by using immunocytochemistry (ICC), a quantitative real time-polymerase chain reaction (qRT-PCR), and a viability assay.

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a protocol to acquire the degenerative tenocyte from humans

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical activity and longer life expectancy. Despite its increasing prevalence, the underlying pathogenesis still remains unclear, and treatment is generally symptomatic. Recently, numerous therapeutic options, including growth factors, stem cells, and gene therapy, were investigated in hopes of enhancing the healing potency of the degenerative tendon. However, the majority of these research studies were conducted only on animal models or healthy human tenocytes. Despite some studies using pathological tenocytes, to the best of our knowledge there is currently no protocol describing how to obtain human degenerative tenocytes. The aim of this study is to describe a standard protocol for acquiring human degenerative tenocytes. Initially, the tendon tissue was harvested from a patient with lateral epicondylitis during surgery. Then biopsy samples were taken from the extensor carpi radialis brevis tendon corresponding to structural changes observed at the time of surgery. All of the harvested tendons appeared to be dull, gray, friable, and edematous, which made them visually distinct from the healthy ones. Tenocytes were cultured and used for experiments. Meanwhile, half of the harvested tissues were analyzed histologically, and it was shown that they shared the same key features of tendinopathy (angiofibroblastic dysplasia or hyperplasia). A secondary analysis by immunocytochemistry confirmed that the cultured cells were tenocytes with the majority of the cells having positive stains for mohawk and tenomodulin proteins. The qualities of the degenerative nature of tenocytes were then determined by comparing the cells with the healthy control using a proliferation assay or qRT-PCR. The degenerative tenocyte displayed a higher proliferation rate and similar gene expression patterns of tendinopathy that matched previous reports. Overall, this new protocol might provide a useful tool for future studies of tendinopathy.

Histological analyses revealed that the harvested tissue from lateral epicondylitis had the characteristics of a tendinopathic tendon.H&#38;E section of tendinopathy degenerative tendon revealed a disorganized collagen bundle with a loss of polarity and fine straight, strongly packed parallel fiber structures. Histological signs suggestive of degeneration such as higher cellularity and enlarged nuclei without the typical spindle shape were common in samples. Additionally, collagen bundles...

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A Protocol to Acquire the Degenerative Tenocyte from Humans

In vitro use of degenerative tenocytes is essential when investigating the efficacy of novel treatment on tendinopathy. However, most research studies use only the animal model or a healthy tenocyte. We propose the following protocol to isolate human degenerative tenocytes during surgery.

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

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Research

JoVE Journal

Medicine

7.3K Views.  CHA University School of Medicine. In vitro use of degenerative tenocytes is essential when investigating the efficacy of novel treatment on tendinopathy. However, most research studies use only the animal model or a healthy tenocyte. We propose the following protocol to isolate human degenerative tenocytes during surgery.

Video: A Protocol to Acquire the Degenerative Tenocyte from Humans

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Developmental Biology

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Genetics

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

Harnessing iPSC-Derived iTenocytes for Effective Cell-Based Therapy

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

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.

Author Spotlight: Advancements in Cell and Tissue Engineering for Tendon Repair 

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.

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

Bioengineering

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker &#945;-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...