Paper Review!

Human umbilical cord stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury

Background

In recent years, it has been discovered that human umbilical cord blood (hUCB) is a valuable source of stem cells, and many studies have looked into its therapeutic potential to initiate and maintain tissue repair. This study investigated the effect of these hUCB mesenchymal stem cells on matrix metalloproteins (MMPs) in rats after spinal cord injury (SCI). MMPs are a large family of proteolytic enzymes involved in inflammation, wound healing, and other pathological processes after neurological disorders, with some being harmful and others beneficial. Although this study looked at a large number of MMPs, its main focus was on MMP-2 which is involved in glial scar formation.

Part of this study was carried out in vivo, using adult male rats of similar weights that were divided into three groups, a sham control group (no injury), a group that were subjected to spinal cord injuries with no treatment, and a third group that were also injured but were injected with hUCB treatment 21 days following the injury. Segments of spinal cord from various rats in each group were removed and analyzed at days 1, 3, 7, and 21.

The second part of this study prepared cultures of spinal neurons (collected from other rats), to test the effect of hUCB on these neurons in vitro. Cultures of spinal neurons were prepared, as well as a coculture of spinal neurons and hUCB. These were exposed to STP, a substance which induces apoptosis, as well as H2O2 which causes free radical damage, because apoptosis and free radical damage are important processes involved in secondary degeneration after SCI. The coculture of spinal neurons and hUCB was also tested with STP in the presence of MMP-2 antibody which blocks the activity of MMP-2.

Results

Real-time PCR
Fold changes of each MMP family member were evaluated in both experimental and control groups using real-time PCR, and it was found that MMP-2 showed only slight upregulation during the 21 days post injury, however when treated with hUCB the upregulation increased substantially. This did not occur in any of the other MMPs (Figure 1).

Figure 1 (click image to enlarge)

MMP-2 activity & expression increased in vivo
Analysis of spinal cords using gelatin zymography showed that bands corresponding to MMP-2 activity appeared most strongly after hUCB treatment, which did not occur with other MMPs (Figure 2).

Figure 2 (click image to enlarge)

The upregulation of MMP-2 by hUCB was further confirmed by immunohistochemical analysis which showed immunoreactivity for MMP-2 was much more prominent in hUCB treated rats compared to control or untreated injured rats where it was hardly detectable. Even further confirmation was provided by western blot analysis, where neurons of spinal cord sections from the hUCB treated rats, taken from an area next to the injury site showed significant expression of MMP-2 compared to injured rats. This suggests that hUCB treatment after SCI causes MMP-2 to be released from neurons next to the injury site (Figure 3).

Figure 3 (click image to enlarge)

hUCB treatment reduced glial scarring
Treatment with hUCB after SCI extensively reduced the amount of glial scarring after injury, while untreated rats showed much more prominent scarring. Glial scars are formed by aggregations of reactive astrocytes in response to CNS injury, interacting with molecules (CSPGs) that arrest regrowth of injured axons across the lesion site. MMP-2 degrades CSPG’s in vitro, allowing axonal regeneration. The hUCB treatment was correlated with both an increase in MMP-2 and a decrease in CSPGs (Figure 4).

Figure 4 (click image to enlarge)

MMP-2 activity & expression increased in vitro
Results of cell survival and cytotoxicity in the in vitro experiment showed that treatment of spinal neurons with either H2O2 and/or STP significantly injured the neurons. This effect of injury was reversed when the neurons were treated with hUCB. When MMP-2 antibody was present, the protection provided by hUCB treatment was significantly reduced, offering further support that MMP-2 is involved in the protection offered by hUCB. These findings were further supported by analysis with gelatin zymography and immunocytochemical analysis (Figures 5 & 6).

Figure 5 (click image to enlarge) Figure 6 (click image to enlarge)

Conclusion
According to the results, it appears that hUCB plays an important role in upregulation of MMP-2 which reduces glial scar formation following spinal cord injury, creating an environment suitable for axonal regeneration. This provides evidence for the therapeutic potential of hUCB treatment for patients suffering from spinal cord injury.

My opinion
The authors of this paper succeeded in presenting a lot of information in a well organized manner that made it easy to read and understand. The experiments were conducted very carefully and analyzed by a variety of methods in order to ensure accurate results. Both experimental and control results were compared and presented clearly, and I especially found the figures very useful. As the authors suggest, future experiments testing transgenic mice or pharmacological inhibitors of specific MMPs would provide further evidence on the therapeutic efficacy of hUCB after spinal cord injury.

Reference
Veeravallia et al., 2009. Human umbilical cord blood stem cells upregulate matrix metalloproteinase-2 in rats after spinal cord injury. Neurobiology of Disease. Volume 36: 200-212 (Link)

My Favourite Tissue! - The Umbilical Cord

Basic Structure and Function

The umbilical cord is a cord that connects the placenta to the fetus during pregnancy, allowing transfer of nutrients and other material between the mother and fetus. Its full length is usually 50-60 cm in length and the diameter generally increases with gestational age [1,6]. It is usually twisted in a spiral shape in either a clockwise or counterclockwise direction, however counterclockwise has been found to be more common[1]. The cord contains three large blood vessels: two arteries that carry deoxygenated blood from the fetus to the placenta, and one vein that carries oxygenated blood to the fetus in the opposite direction. These are embedded in a mucous connective tissue known as Wharton’s Jelly. The umbilical cord does not contain lymphatic vessels or nerve fibers[1,5].
* Although the general agreement is that there are no nerve
fibers in the cord, there have been some studies that have found different results[1].



Development of the Umbilical Cord

The umbilical cord begins to develop with the formation of an extraembryonic coelom that almost surrounds the early embryo and also remains attached to the chorion by a connecting stalk of mesenchyme, which later becomes ventrally located during development. The amniotic sac expands as the embryo grows, filling the extraembryonic coelom and compressing the remnants of both the yolk sac and a duct called the vitello-intestinal duct, against the connecting stalk. Eventually these structures all fuse to form the umbilical cord, which is surrounded by the amnion and amniotic cavity at this point. By the middle of the 5th month of gestation, many of these structures disappear, and all that remains are two umbilical arteries and a single umbilical vein embedded in the Wharton’s Jelly, which consists mainly of ground substance[3].
Figure 2: Development of the umbilical cord (click here to enlarge figure)


Histology

Epithelium
The umbilical cord is covered in amniotic epithelium that varies along the length of the cord. The area near the umbilicus (navel), is covered in an unkeratinized, stratified squamous epithelium which provides the transition from the abdominal wall to the cord surface. As the distance increases away from the naval, the epithelium changes into 2 to 8 layers of stratified columnar epithelium, and finally into a simple columnar epithelium. Unlike the amniotic surface of the placenta, the amnion of the cord grows firmly into the central connective tissue core and cannot be moved[1].


Wharton's jelly
The bulk of the umbilical cord is comprised of mucous connective tissue known as Wharton’s Jelly, shown in the figures below. It contains ground substance rich in GAG’s, primarily hyaluronic acid, as well as fibroblasts and a delicate network of collagenous fibers[1,4]. The extracellular matrix is hydrophilic giving the jelly-like consistency. The fibroblasts are spindle shaped and evenly distributed with long extensions, and there are numerous mast cells present that surround the vessels and are also found beneath the cord surface. Immunohistochemically, the interstitial collagens types I, III, and VI, as well as the basal lamina molecules collagen type IV, laminin and heparin sulphate have all been found[1].














According to certain studies, stromal cells in Wharton’s Jelly show different degrees of differentiation from mesenchymal cells to myofibroblasts depending on their location. The most immature cells are close to the amniotic surface and still proliferating, however, with increasing distance of the amniotic surface, the cells acquire cytoskeletal features of contractile cells. The stromal cells close to the umbilical vessels were found to be highly differentiated myofibroblasts[1].

Pathology

Umbilical cord problems account for 9% of fetal deaths, and are related to a large variety of abnormalities and other negative consequences. There are numerous types of problems that may occur with the umbilical cord, however not all of their causes and consequences are fully understood. The following are brief descriptions about some of the problems that may occur.

Short Cord Length
A cord is considered short when is has a total length of 40 cm or less, although they are relatively uncommon. Those less than 15 cm are often associated with abdominal wall defects, as well as spinal and limb deformities. Short cords are correlated with depressed IQ, and have been found in offspring who suffer from Fetal Alcohol syndrome as well as Down’s syndrome.

Long Cord Length
Long cords are typically those that have a total length of over 70 cm and have been found to be associated with a significant increase in risk of brain abnormalities and/or abnormal neurological function. Knots and fatal strictures are nearly always found with cords of excessive length. Also, long cords may prolapsed or become entangled around itself or the fetus. When a cord loops around a fetus’s neck, it is referred to as a nuchal cord loop. This occurs in 20-33% of normal term pregnancies, is slightly more common in males, and includes 2 types:
Type A: The cord encircles the neck in an unlocked pattern
Type B: The cord encircles the neck in a locked pattern
The latter type is usually associated with more severe consequences, however for the most part, nuchal cords do not affect the outcome of the pregnancy or the fetal weight at birth.

Knots
Fetuses with knots in their umbilical cords are at a 4-fold risk of intrauterine death compare to those with normal cords. Knots may cause compression of Wharton’s jelly, lead to significant prepartum hypoxia with lasting damage, or cause death. There are 2 types of knots:
True knots – These are associated with thrombosis of placental surface veins and an increase in
still births (figure 7).
False Knots – These may be large, but are named poorly, as they are actually local redundancies
of umbilical vessels rather than knots (figure 8).


Figure 7: True Knot [1]



Abnormal cord insertion
Furcate cord Insertion – Occasionally a rare abnormality occurs in which the umbilical vessels separate from the cord substance prior to reaching the surface of the placenta, therefore losing the protection of the Wharton’s jelly. This greatly increases occurrences of thrombosis and injury.

Velementous cord insertion – This occurs when the insertion of the umbilical cord is in the membrane of the placenta rather than within the placenta, and may be close to the placenta itself or farther away at the apex of the membrane. It is common in multiple births, and those where IUD’s are found in placental membranes. Haemorrhages are the more frequent complication of these membranous vessels.

Cysts and Edema
Cysts in Whartons jelly are rare but occur, and are most obvious in edementous umbilical cords that have been associated with some cases of respiratory distress syndrome of newborns. Usually, cysts are not accompanied by fetal disease.
Edema is apparent in the markedly swollen, glistening umbilical cord.

Strictures
Strictures are significant reductions in the size of the umbilical cord and are not uncommon. They are found in long, heavy spiralled cords, and although fetal grasping or knots may cause them, the primary cause is a deficiency of Wharton’s jelly.

Hematomas
Umbilical cord hematomas are associated with 50% of fetal mortality. Some studies have showed that short cords, trauma, and entangling play a role in the formation of a hematoma, but the primary cause remains generally unknown[1].

Interesting Facts

* Wharton’s Jelly liquefies when touched[1]

* Leonardo da Vinci suggested that the length of the umbilical cord is about the same as the length of the baby, which has been supported by many current studies[1].

* A 2006 study found that infants who had their umbilical cord clamped immediately after birth had much lower iron levels later in life than those who had a two minute delay in the clamping of their umbilical cord. The delay increased iron stores by about 27-47mg, suggesting that delaying cord clamping could help prevent iron deficiency from developing early in life[2].

References

Baergen, Rebecca; Kurt Benirschke, Peter Kaufmann. (2006). Pathology of the human placenta, 5th edition. Springer New York. pp. 380-451 [1]

Chapero, C; L. Neufeld, G. Tena Alavez, R. Eguia-Liz Cedillo, K. Dewey. (2006). Effect of timing of umbilical cord clamping on iron status in mexican infants: a randomised controlled trial. Lancet. (9527) pp 1997-2004 [2]

Heath, John W.; James S. Lowe, Alan Stevens, Barbara Young. (2006). Wheater's functional histology: A text and colour atlas, 5th edition. Elsevier. p 385 [3]

Junqueira, Luiz Carlos; Jose Carneiro. (2005). Basic Histology: Text and atlas, 11th edition. McGraw-Hill companies. [4]

Meyer, David B; (1985). Laboratory guide for human histology, revised edition. Wayne State University Press. p 76 [5]

Zhang, Shu-Xin. (1999). An atlas of histology. Springer, New York, Berlin. pp 334-336[6]