Making Cartilage from Umbilical Cord Stem Cells Without Growth Factors


Loïc Reppel and his colleagues at CNRS-Université de Lorraine in France have found that mesenchymal stem cells from human umbilical cord can not only be induced to make cartilage, but that these remarkable cells can make cartilage without the use of exogenous growth factors.

Mesenchymal stromal/stem cells from bone marrow (BM-MSC) have, for some time, been the “all stars” for cartilage regeneration. In fact, a very innovative clinic near Denver, CO has pioneered the use of BM-MSCs for patients with cartilage injuries. Chris Centeno, the mover and shaker, of this clinic has carefully documented the restoration of articular cartilage in many patients in peer-reviewed articles.

However, there is another “kid’ on the cartilage-regeneration block; mesenchymal stromal/stem cells from Wharton’s jelly (WJ-MSC). The advantages of these cells are their low immunogenicity and large cartilage-making potential. In this paper, which was published in Stem Cell Research and Therapy, Reppel and others evaluated the ability of WJ-MSCs to make cartilage in three-dimensional culture systems.

Reppell and his coworkers embedded WJ-MSCs isolated from the umbilical cords of new-born babies in alginate/hyaluronic acid hydrogel and grew them for over 28 days. These hydrogels were constructed by the spraying method. The hydrogel solution (for those who are interested, it was 1.5 % (m/v) alginate and hyaluronic acid (ratio 4:1) dissolved in 0.9 % NaCl) was sprayed an airbrush connected to a compressor. The solution was seeded with WJ-MSCs and then sprayed on a sterile glass plate. The hydrogel was made solid (gelation) in a CaCl 2 bath (102 mM for 10 minutes). Then small cylinders were cut (5 mm diameter and 2 mm thickness) with a biopsy punch. Then Reppel and others compared the chondrogenic differentiation of WJ-MSC in these three-dimensional scaffolds, without adding growth factors with BM-MSC.

Illustration of protocol steps used to perform scaffold construct and chondrogenic differentiation. After monolayer expansion, MSC were seeded at 3× 10 6 cells/mL of Alg/HA hydrogel. Hydrogel was sprayed, gelated, and cut into 5 mm diameter cylinders; scale bar = 5 mm. Scaffolds were cultivated in a 48-well plate in differentiation medium for 28 days. Alg/HA alginate/hyaluronic acid, MSC mesenchymal stromal/stem cells, P3 passage 3
Illustration of protocol steps used to perform scaffold construct and chondrogenic differentiation. After monolayer expansion, MSC were seeded at 3× 10 6 cells/mL of Alg/HA hydrogel. Hydrogel was sprayed, gelated, and cut into 5 mm diameter cylinders; scale bar = 5 mm. Scaffolds were cultivated in a 48-well plate in differentiation medium for 28 days. Alg/HA alginate/hyaluronic acid, MSC mesenchymal stromal/stem cells, P3 passage 3

After 3 days in culture, WJ-MSCs seemed nicely adapted to their new three-dimensional culture system without any detectable damage. From day 14 – 28, the proportion of WJ-MSC cells that expressed all kinds of cell surface proteins characteristic of MSCs (i.e., CD73, CD90, CD105, and CD166) decreased significantly. This suggests that these cells were differentiating into some other cell type.

After 28 days in this scaffold culture, both WJ-MSCs and BM-MSCs showed strong upregulation of cartilage-specific genes. However, WJ-MSCs exhibited greater type II collagen synthesis than BM-MSCs, and these differences were evident at the RNA and protein levels. Collagen II is a very important molecule when it comes to cartilage synthesis because chondrogenesis, otherwise known as cartilage production, occurs when MSCs differentiate into cartilage-making cells known as chondroblasts that begins secreting aggrecan and collagen type II that form the extracellular matrix that forms cartilage. Unfortunately, in order to complete the run to mature cartilage formation, the chrondrocytes must enlarge (hypertrophy), and express the transcription factor Runx2 and secrete collagen X. Unfortunately, WJ-MSCs expressed Runx2 and type X collagen at lower levels than BM-MSCs in this culture system.

Matrix synthesis detected after 28 days of chondrogenic induction. Proteoglycans and total collagen were stained by Alcian blue and Sirius red (a), respectively. To explore the synthesis of various collagens in depth, immunofluorescence (b) and immunohistochemistry staining (c) were performed and detected using fluorescence microscopy and light microscopy, respectively; scale bar = 100 μm. BM-MSC bone marrow-derived mesenchymal stromal/stem cells, WJ-MSC Wharton’s jelly-derived mesenchymal stromal/stem cells
Matrix synthesis detected after 28 days of chondrogenic induction. Proteoglycans and total collagen were stained by Alcian blue and Sirius red (a), respectively. To explore the synthesis of various collagens in depth, immunofluorescence (b) and immunohistochemistry staining (c) were performed and detected using fluorescence microscopy and light microscopy, respectively; scale bar = 100 μm. BM-MSC bone marrow-derived mesenchymal stromal/stem cells, WJ-MSC Wharton’s jelly-derived mesenchymal stromal/stem cells

These experiments only examined cells in culture, which is not the same as placing cells in a living animal, but it is a start. Thus, when they are seeded in the hydrogel scaffold, WJ-MSCs and BM-MSCs, after 4 weeks, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins in the absence of growth factors. In order to properly make cartilage in clinical applications, WJ-MSCs must go the full way and express high levels of Runx2 and collagen X. However, these experiments show that WJ-MSCs, which in the past were medical waste, are a potential alternative source of stem cells for cartilage tissue engineering.

Reppel and his colleagues note in their paper that to improve cartilage production from WJ-MSCs, it might be important to mimic the physiological environment in which chondrocytes normally find themselves. For example, they could apply mechanical stress or even a low-oxygen culture system. Additionally, Reppel and others could apply stratified cartilage tissue engineering. Reppel thinks that they could adapt their spraying method to design new stratified engineered tissues by applying progressive cells and spraying hydrogel layers one at a time.

All in all, cartilage repair based with WJ-MSC embedded in Alginate/Hyaluronic Acid hydrogel will hopefully be tested in laboratory animals and then, perhaps, if all goes well, in clinical trials.

Advertisements

Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).