Mesenchymal stem cell Protocols | Adult stem cell | Stem Cell

A Protocol to Induce Brown and Beige Adipocyte Differentiation From Murine and Human Adipose-Derived SVF

RY Rohit Raj Yadav

NM Nandita Mishra

NV Narendra Verma

1886 Views

Dec 5, 2025

Adipose cells vary functionally, with white adipocytes storing energy and brown/beige adipocytes generating heat. Mouse and human subcutaneous white adipose tissue (WAT)-derived stromal vascular fraction (SVF) provides mesenchymal stem cells (MSCs) that can be differentiated into thermogenic adipocytes using pharmacological cocktails. After six days of browning induction, these cells exhibited significant upregulation of thermogenic markers (UCP1, Cidea, Dio2, PRDM16) along with adipogenic genes (PPARγ, aP2), showing enhanced thermogenic potential. This in vitro system offers a practical platform to study adipogenesis and thermogenic regulation.

Spheroid Sheets: A Scalable Platform for Producing Tissue Membrane Constructs

QL Quang Bach Le

HE Hariharan Ezhilarasu

WC Weng Wan Chan

DC Deepak Choudhury

0 Q&A

1716 Views

Nov 20, 2025

Bottom-up tissue engineering using cell spheroids offers many advantages in recapitulating native cell–cell and cell–matrix interactions. Many tissues, such as cartilage, bone, cardiac muscle, intestine, and neural tissues, have been tissue-engineered using cell spheroids. However, previous methods for spheroid assembling, such as mold casting, hydrogel-based bioprinting, or needle array, either lack control over final tissue geometry or face challenges in scalability and throughput. In this protocol, we describe a robust and scalable tissue engineering method for assembling cell spheroids into a thin, planar spheroid sheet. The spheroids are sandwiched between two flexible meshes held by a frame, facilitating uniform spheroid fusion while ensuring nutrient exchange and ease of handling. We demonstrate this method by producing thin cartilage tissue from human mesenchymal stem cells undergoing chondrogenic differentiation. This approach offers a practical platform for producing thin membrane-like tissue constructs for many research and therapeutic applications.

A Full Good Manufacturing Practice–Compliant Protocol for Corneal Stromal Stem Cell Cultivation

MS Mithun Santra

YH Yen-Michael S. Hsu

SK Shaheen Khadem

SR Sydney Radencic

MF Martha L. Funderburgh

OS Onkar B. Sawant [...]

GY Gary H.F. Yam

+ 2 Authors

0 Q&A

2308 Views

Sep 20, 2024

Corneal scarring, a significant cause of global blindness, results from various insults, including trauma, infections, and genetic disorders. The conventional treatment to replace scarred corneal tissues includes partial or full-thickness corneal transplantation using healthy donor corneas. However, only 1 in 70 individuals with treatable corneal scarring can undergo surgery, due to the limited supply of transplantable donor tissue. Our research focuses on cell-based strategies, specifically ex vivo–expanded corneal stromal stem cells (CSSCs), to address corneal scarring. Preclinical studies have demonstrated the efficacy of CSSC treatment in reducing corneal inflammation and fibrosis, inhibiting scar formation, and regenerating native stromal tissue. Mechanisms include CSSC differentiation into stromal keratocytes and the expression of regenerative cytokines. Here, we present a good manufacturing practice (GMP)-compliant protocol to isolate and expand human CSSCs. This method paves the way to produce clinical-grade CSSCs for transplantation and clinical trials.

Isolation and ex vivo Expansion of Limbal Mesenchymal Stromal Cells

NP Naresh Polisetti

LS Lyne Sharaf

TR Thomas Reinhard

GS Günther Schlunck

0 Q&A

3111 Views

Jul 20, 2022

Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic.

Graphical abstract:

Isolation of Extracellular Vesicles Derived from Mesenchymal Stromal Cells by Ultracentrifugation

María José Ramírez-Bajo

Elisenda Banon-Maneus

Jordi Rovira

Josep M. Campistol

Fritz Diekmann

1 Q&A

6625 Views

Dec 20, 2020

Extracellular vesicles (EVs) are a heterogeneous group of membranous vesicles that differ on their biogenesis and release pathways, such as exosomes, microvesicles and apoptotic bodies. They are involved in cell-to-cell communication delivering signal molecules (proteins, nucleic acids, lipids, etc.) that can regulate different physiological processes, as well as the development and progression of several diseases. There are different methods and commercial kits to isolate EVs and depending on the methodology one could obtain EVs with different degrees of efficiency, purity and it can be more or less time-consuming. Then, the choice has to be according to the different advantages and disadvantages, and their use for downstream applications. Here, we describe the EVs isolation method from mesenchymal stromal cells by ultracentrifugation. This EVs isolation can be performed using common media and buffers, and only with the requirement of an analytical ultracentrifuge. Moreover, this method can be used to obtain large quantity of EVs with a good reproducibility for developing in vitro and in vivo experiments and studying their biological actions.

Cell Microencapsulation and Cryopreservation with Low Molecular Weight Hyaluronan and Dimethyl Sulfoxide

HG H. Gurruchaga

LS L. Saenz del Burgo

GO G. Orive

RH R. M. Hernandez

J. Ciriza

JP J. L. Pedraz

0 Q&A

6475 Views

Feb 20, 2019

Cryopreservation is commonly used for the storage of cells, tissues, organs or 3D cell-based products using ultra-low temperatures, which involves the immersion in liquid nitrogen for their long-term preservation. The cryopreservation of several microencapsulated cells is usually performed by the slow freezing with the dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA). In this study, we cryopreserved several microencapsulated cells with the natural, non-toxic low molecular-weight hyaluronan (LMW-HA) at 5% and DMSO 10% solution assessing cell viability and metabolic activity after thawing. The cryopreservation of microencapsulated D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) with the LMW-HA 5% presented similar outcomes after thawing compared to the DMSO solution, showing the low molecular weight hyaluronan as a natural, non-toxic CPA that can be used preventing the DMSO related adverse effects after the implantation of the cryopreserved cell-based products.

Isolation of Multipotent Mesenchymal Stem Cells from Human Extraocular Muscle Tissue

AS Amit Sharma

DM Darilang Mawrie

DM Damarais Magdalene

BJ Bithiah Grace Jaganathan

0 Q&A

7489 Views

Feb 20, 2019

Mesenchymal stem cells (MSCs) have attracted significant attention as potential therapeutic cells to treat various diseases ranging from tissue injuries, graft versus host disease, degenerative diseases and cancer. Since the initial discovery of MSCs in the bone marrow cells, MSCs have been successfully isolated from various adult and neo-natal tissues, albeit the procedures are often coupled with difficulties in harvesting tissue and produce low yield of cells, requiring extensive expansion in vitro. Here, we explored extra-ocular muscle tissues obtained from patients as a novel source of MSCs which express characteristic cell surface markers of MSCs and show multilineage differentiation potential with high proliferation capacity.

One-step Derivation of Functional Mesenchymal Stem Cells from Human Pluripotent Stem Cells

0 Q&A

10188 Views

Nov 20, 2018

Mesenchymal stem cells (MSCs) are invaluable cell sources for understanding stem cell biology and potential application in tissue engineering and regenerative medicine. The current issues of MSCs that demand to be further addressed are limited donors, tissue sources and limited capacity of ex vivo expansion. Here, we describe a simple and easy protocol for generating functional mesenchymal stem cells from human pluripotent stem cells (hPSCs) via one-step low glucose medium switch strategy in feeder-free culture system. In this protocol, human induced pluripotent stem cells (hiPSCs) and H9 human embryonic stem cells (hESCs) were successfully differentiated into MSCs, named hiPSC-MSCs and hESC-MSCs, respectively. The derived hiPSC-MSCs and hESC-MSCs exhibited common MSC characteristics as MSCs derived from human bone marrow (hBM-MSCs), including expressing MSC surface markers and possessing capability of tri-lineage differentiation in vitro (adipogenesis, osteogenesis and chondrogenesis). As compared with other available protocols, our protocol can be applied to generate a large number of MSCs from hPSCs with high efficiency, low-cost manner, moreover, not involving embryoid body, mouse feeder-cell, flow sorting, and pathway inhibitors (such as SB203580 and SB431542). We believe that this protocol could provide a robust platform to reach the future demand for producing the industrial scale of MSC from hPSCs for autologous cell-based therapy.

Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates

Elena Redondo-Castro

Catriona J Cunningham

Jonjo Miller

Stuart A Cain

SA Stuart M Allan

Emmanuel Pinteaux

0 Q&A

9961 Views

Aug 20, 2018

The 3D culture of human mesenchymal stem cells (hMSCs) represents a more physiological environment than classical 2D culture and has been used to enhance the MSC secretome or extend cell survival after transplantation. Here we describe a simple and affordable method to generate 3D spheroids of hMSCs by seeding them at high density in a low-binding 96-well plate.

Spheroids of hMSCs cultured in low-binding 96-well plates can be used to study the basic biology of the cells and to generate conditioned media or spheroids to be used in transplantation therapeutic approaches. These MSCs or their secretome can be used as a regenerative therapy and for tissue repair across multiple disease areas, including neurodegeneration.

In comparison to other methods (hanging drop, use of gels or biomaterials, magnetic levitation, etc.), the method described here is simple and affordable with no need to use specialized equipment, expensive materials or complex reagents.

Measurement of Mesenchymal Stem Cells Attachment to Endothelial Cells

SW Shan Wang

CM Chris D. Madsen

YW Yaojiong Wu

1 Q&A

8317 Views

Mar 20, 2018

Mesenchymal stem cells (MSCs) have shown profound therapeutic potential in tissue repair and regeneration. However, recent studies indicate that MSCs are largely entrapped in lungs after intravenous delivery and die shortly. The underlying mechanisms have been poorly understood. We have provided evidence to show that excess expression and activation of integrins in culture-expanded MSCs is a critical cause of MSCs adhesion to endothelial cells of the lung microarteries resulting in the entrapment of the cells (Wang et al., 2015). Therefore, it may be meaningful to test the adhesive ability of MSCs to endothelial cells in vitro before intravenous administration to avoid their lung vascular obstructions. Here we report a simple method to measure MSCs attachment to endothelial cells.