Amniotic Fluid Stem Cells Make Robust Blood Vessel Networks


The growth of new blood vessels in culture received in new boost from researchers at Rice University and Texas Children’s Hospital who used stem cells from amniotic fluid to promote the growth of robust, functional blood vessels in healing hydrogels.

These results were published in the Journal of Biomedical Materials Research Part A.

Engineer Jeffrey Jacot thinks that amniotic fluid stem cells are valuable for regenerative medicine because of their ability to differentiate into many other types of cells, including endothelial cells that form blood vessels. Amniotic fluid stem cells are taken from the discarded membranes in which babies are encased in before birth. Jacot and others combined these cells with an injectable hydrogel that acted as a scaffold.

In previous experiments, Jacot and his colleagues used amniotic fluid cells from pregnant women to help heal infants born with congenital heart defects. Amniotic fluids, drawn during standard tests, are generally discarded but show promise for implants made from a baby’s own genetically matched material.

“The main thing we’ve figured out is how to get a vascularized device: laboratory-grown tissue that is made entirely from amniotic fluid cells,” Jacot said. “We showed it’s possible to use only cells derived from amniotic fluid.”

Researchers from Rice, Texas Children’s Hospital and Baylor College of Medicine combined amniotic fluid stem cells with a hydrogel made from polyethylene glycol and fibrin. Fibrin is the proteins formed during blood clots, but it is also used for cellular-matrix interactions, wound healing and angiogenesis (the process by which new vessels are made). Fibrin is widely used as a bioscaffold but it suffers from low mechanical stiffness and is degraded rapidly in the body. When fibrin was combined with polyethylene glycol, the hydrogel became much more robust, according to Jacot.

Additionally, these groups used a growth factor called vascular endothelial growth factor to induce the stem cells to differentiate into endothelial cells. Furthermore, when induced in the presence of fibrin, these cells infiltrated the native vasculature from neighboring tissue to make additional blood vessels.

When mice were injected with fibrin-only hydrogels, thin fibril structures formed. However if those same hydrogels were infused with amniotic fluid stem cells that had been induced with vascular endothelial growth factor, the cell/fibrin hydrogel concoctions showed far more robust vasculature.

In similar experiments with hydrogels seeded with bone marrow-derived mesenchymal cells, once again, vascular growth was observed, but these vessels did not have the guarantee of a tissue match. Interestingly, seeding with endothelial cells didn’t work as well as the researchers expected, he said.

Jacot and others will continue to study the use of amniotic stem cells to build biocompatible patches for the hearts of infants born with birth defects and for other procedures.

University of Pittsburgh Team Uses Patient’s Own Stem Cells to Clear Cloudy Corneas


The transparent portion of the center of our eyes is called the cornea. Scars on the cornea can cause an infuriating haziness across the eye. However, healing these cloudy corneas might be as simple as growing stem cells from a tiny biopsy of the patient’s undamaged eye and placing them on the injury site. This hope comes from experiments in a mouse model system conducted by researchers at the University of Pittsburgh School of Medicine. These findings were published in Science Translational Medicine and could one day rescue vision for millions of people worldwide and decrease the need for corneal transplants.

According to statistics compiled by the National Eye Institute, which is a branch of the National Institutes of Health, globally, corneal infectious diseases have compromised the vision of more than 250 million people and have blinded over 6 million of them. Additionally, trauma from burns is also a leading cause of corneal scarring.

James L. Funderburgh, Ph.D., professor of ophthalmology at Pitt and associate director of the Louis J. Fox Center for Vision Restoration of UPMC and the University of Pittsburgh, a joint program of UPMC Eye Center and the McGowan Institute for Regenerative Medicine, said, “The cornea is a living window to the world, and damage to it leads to cloudiness or haziness that makes it hard or impossible to see. The body usually responds to corneal injuries by making scar tissue. We found that delivery of stem cells initiates regeneration of healthy corneal tissue rather than scar leaving a clear, smooth surface.”

The lead author of this study, Sayan Basu, is a corneal surgeon who works at the L.V. Prasad Eye Institute in Hyderabad, India. Dr. Basu who joined with Dr. Funderburgh’s lab, has developed a technique to isolate ocular stem cells from tiny biopsies from the surface of the eye and a region between the cornea and sclera known as the limbus. Such a small biopsy heals rapidly with little discomfort and no disruption of vision. Such biopsies are banked in tissue banks and then expanded in culture, and several tests shows that even after isolation and expansion, these cells are still corneal stem cells.

limbal-stem-cells

“Using the patient’s own cells from the uninjured eye for this process could let us bypass rejection concerns,” Dr. Basu noted. “That could be very helpful, particularly in places that don’t have corneal tissue banks for transplant.”

Basu in collaboration with Funderburgh’s team tested these human limbal stem cells in a mouse model of corneal injury. This team used goo made of fibrin to glue the cells to the injury site. Fibrin is the protein found in blood clots, but it is also commonly used as a surgical adhesive. Application of these limbal stem cells not only induced healing of the mouse corneas, their eyes became clear again within four weeks of treatment. On the other hand, the eyes of mice that were not treated with limbal stem cells remained cloudy.

Fibrin

In fact, the healing was so good that Funderburgh said: “Even at the microscopic level, we couldn’t tell the difference between the tissues that were treated with stem cells and undamaged cornea. We were also excited to see that the stem cells appeared to induce healing beyond the immediate vicinity of where they were placed. That suggests the cells are producing factors that promote regeneration, not just replacing lost tissue.”

This work is the impetus behind a small pilot study presently underway in Hyderabad which will treat a handful of patients with their own corneal stem cells.

Artificial Skin Created Using Umbilical Cord Stem Cells


Major burn patients usually must wait weeks for artificial skin to be grown in the laboratory to replace their damaged skin, buy a Spanish laboratory has developed new protocols and techniques that accelerate the growth of artificial skin from umbilical cord stem cells. Such laboratory-grown skin can be frozen and stored in tissue banks and used when needed.

Growing skin in the laboratory requires the acquisition of keratinocytes, those cells that compose the skin and the mucosal covering inside our mouths.  Keratinocytes can be cultured in the laboratory, but they have a long cell cycle, which means that they take a really long time to divide.  Consequently, cell cultures of keratinocytes tend to take a very long time to grow.

Keratinocytes in culture
Keratinocytes in culture

As they grow, the keratinocytes respond to connective tissue underneath them to receive the cues that tell them how to connect with each other and form either skin or oral mucosa.  In patients with severe burns, however, the underlying connective tissue is also often damaged.  Therefore, finding a way to not only accelerate the growth of cultured keratinocytes, but also to provide the underlying structure that directs the cells to form a proper epithelium is essential.

Remember that severe burn patients are living on borrowed time.  Without a proper skin covering, water loss is severe and dehydration is a genuine threat.  Also, infection is another looming threat.  Therefore, the treatment of a burn patient is a race against time.

Because umbilical cord stem cells grow quickly and effectively in culture, they might be able to differentiate into keratinocytes and form the structures associated with oral mucosa and skin.

University of Granada researchers used a new type of epithelial covering to grow their artificial skin in addition to a biomaterial made of fibrin (the stiff, cable-like protein that forms clots) and agarose to provide the underlying connective tissue. In case you might need a refresher, an epithelium refers to a layer of cells that have distinct connects with each other and form a discrete layer. Epithelia can form single or multiple layers and can be composed of long, skinny cells, short, flat cells, or boxy cells.  An epithelium is a membrane-like tissue composed of one or more layers of cells separated by very little intervening substances.  Epithelia cover most internal and external surfaces of the body and its organs.

Previous work from this same research group showed that stem cells from Wharton’s jelly (connective tissue within the umbilical cord), could be converted into epithelial cells. This current study confirms and extends this previous work and applies it to growing skin, and oral mucosa.

“Creating this new type of skin suing stem cells, which can be stored in tissue banks, mains that it can be used instantly when injuries are caused, and which would bring the application of artificial skin forward many weeks,” said Antonio Campos, professor of histology and one of the authors of this study.

By growing the Wharton’s jelly stem cells on their engineered matrix in a three-dimensional culture system, Campos and his colleagues saw that the stem cells stratified (formed layers), and expressed a bunch of genes that are peculiar to skin and other types of epithelia that cover surfaces (e.g., cytokeratins 1, 4, 8, and 13; plakoglobin, filaggrin, and involucrin).  When examined with an electron microscope, the cells had truly formed the kinds of tight connections and junctions that are so common to skin epithelia.

Electron microscopy analysis of controls and three-dimensional bioactive models of H-hOM and H-hS. SEM images (top) corresponding to N-hOM and N-hS controls showed a tight superficial layer of flat polygonal cells with desquamation signs in which cells were covering the entire surface, whereas samples kept in vitro for 2 weeks showed immature differentiation patterns, and samples implanted in vivo for 40 days tended to resemble the structure of the native control tissues, with flattened cells and evident signs of desquamation. Scale bars = 50 μm. TEM samples (bottom) were analyzed after 40 days of in vivo implantation and demonstrated that in vivo-implanted tissues were mature and well-differentiated, with numerous intercellular junctions, abundant cell organelles, and a collagen-rich stroma. Scale bars = 1 μm. Abbreviations: H-hOM, heterotypical human oral mucosa; H-hS, heterotypical human skin; N-hOM, native human oral mucosa; N-hS, native human skin; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
Electron microscopy analysis of controls and three-dimensional bioactive models of H-hOM and H-hS. SEM images (top) corresponding to N-hOM and N-hS controls showed a tight superficial layer of flat polygonal cells with desquamation signs in which cells were covering the entire surface, whereas samples kept in vitro for 2 weeks showed immature differentiation patterns, and samples implanted in vivo for 40 days tended to resemble the structure of the native control tissues, with flattened cells and evident signs of desquamation. Scale bars = 50 μm. TEM samples (bottom) were analyzed after 40 days of in vivo implantation and demonstrated that in vivo-implanted tissues were mature and well-differentiated, with numerous intercellular junctions, abundant cell organelles, and a collagen-rich stroma. Scale bars = 1 μm. Abbreviations: H-hOM, heterotypical human oral mucosa; H-hS, heterotypical human skin; N-hOM, native human oral mucosa; N-hS, native human skin; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

The authors conclude the article with this statement: “All these findings support the idea that HWJSCs could be useful for the development of human skin and oral mucosa tissues for clinical use in patients with large skin and oral mucosa injuries.”  Think of it folks – new skin for burn patients, quickly, safely and ethically.

Now back to reality – this is exciting, but it is a a pre-clinical study.  Larger animals studies must show the efficacy and safety of this protocol before human trials can be considered, but you must admit that it looks exciting; and without killing any embryos.

See I. Garzón, et al., Stem Cells Trans MedAugust 2013 vol. 2 no. 8625-632.