Induced Pluripotent Stem Cells from Diabetic Foot Ulcer Fibroblasts


Dr. Jonathan Garlick is professor of Oral Pathology at Tufts University and has achieved some notoriety among stem cell scientists by publishing a stem-cell rap on You Tube to teach people about the importance of stem cells.

Garlick and his colleagues have published a landmark paper in the journal Cellular Reprogramming in which cells from diabetic patients were reprogrammed into induced pluripotent stem cells (iPSCs).

Garlick and his colleagues have established, for the first time, that skin cells from diabetic foot ulcers can be reprogrammed iPSCs. These cells can provide an excellent model system for diabetic wounds and may also used, in the future, to treat chronic wounds.

ESC and iPSCs differentiation to fibroblast fate. ESC and iPSC were differentiated and monitored at various stages of differentiation. Representative images show the morphology of ESC and 2 iPSC lines after days 1, 4, 7, 10, 14, 21 and 28 of differentiation. Early morphologic changes showed differentiation beginning at the periphery of colonies (day 1). At later stages cells acquired fibroblast features of elongated, stellate cells (day 10 at days 21 and 28 of differentiation.
ESC and iPSCs differentiation to fibroblast fate. ESC and iPSC were differentiated and monitored at various stages of differentiation. Representative images show the morphology of ESC and 2 iPSC lines after days 1, 4, 7, 10, 14, 21 and 28 of differentiation. Early morphologic changes showed differentiation beginning at the periphery of colonies (day 1). At later stages cells acquired fibroblast features of elongated, stellate cells (day 10 at days 21 and 28 of differentiation.

Garlick’s team at Tufts University School of Dental Medicine and the Sackler School of Graduate Biomedical Sciences at Tufts, have also used their diabetic-derived iPSCs to show that a protein called fibronectin is linked to a breakdown in the wound-healing process in cells from diabetic foot ulcers.

One of the goals of Garlick’s research is to develop efficient protocols to make functional cell types from iPSCs and to use them to generate 3D tissues that demonstrate a broad range of biological functions. His goal is to use the 3D model system to develop human therapies to replace or regenerate damaged human cells and tissues and restore their normal function.

In this paper, Garlick and his colleagues showed that not only can fibroblasts from diabetic wounds form iPSCs, but they can also participate in 3D skin-like tissues. This model system is more than a disease-in-a-dish system but disease-in-a-tissue system.

Fabrication of three-dimensional tissue construction. (A) A collagen gel embedded with human dermal fibroblasts is layered onto a polycarbonate membrane. (B) After dermal fibroblasts contract and remodel the collagen matrix, keratinocytes are then seeded onto it to create a monolayer that will form the basal layer of the tissue. (C) Tissues are raised to an air-liquid interface to initiate tissue development that mimics in vivo skin.
Fabrication of three-dimensional tissue construction. (A) A collagen gel embedded with human dermal fibroblasts is layered onto a polycarbonate membrane. (B) After dermal fibroblasts contract and remodel the collagen matrix, keratinocytes are then seeded onto it to create a monolayer that will form the basal layer of the tissue. (C) Tissues are raised to an air-liquid interface to initiate tissue development that mimics in vivo skin. From this site.

“The results are encouraging. Unlike cells taken from healthy human skin, cells taken from wounds that don’t heal – like diabetic foot ulcers – are difficult to grow and do not restore normal tissue function,” said Garlick. “By pushing these diabetic wound cells back to this earliest, embryonic stage of development, we have ‘rebooted’ them to a new starting point to hopefully make them into specific cell types that can heal wounds in patients suffering from such wounds.”

Scientists in Garlick’s laboratory used these 3D tissues to test the properties of cells from diabetic foot ulcers and found that cells from the ulcers get are not able to advance beyond synthesizing an immature scaffold made up predominantly of a protein called fibronectin.  Fibronectin, unfortunately, seems to prevent proper closure of wounds.

Fibronectin Sigma

Fibronectin has been shown to be abnormal in other diabetic complications, such as kidney disease, but this is the first study that directly connects it to cells taken from diabetic foot ulcers.

Deriving more effective therapies for foot ulcers has been slow going because of a lack of realistic wound-healing models that mimic the extracellular matrices of human tissues. This scaffolding is critical for wound repair in skin, and other tissue as well.

The work in this paper builds on earlier experiments that showed that cells from diabetic ulcers have fundamental defects that can be simulated using laboratory-grown 3D tissue models. These 3D models will almost certainly be a good model system to test new therapeutics that could improve wound healing and prevent those limb amputations that result when treatments fail.

Garlick’s 3D model will allow him and other researchers to push these studies forward. Can they differentiate their cells into more mature cell types that can be studied in 3D models to see if they will improve healing of chronic wounds?

More than 29 million Americans have diabetes. Diabetic foot ulcers, often resistant to treatment, are a major complication. The National Diabetes Statistics Report of 2014 stated that about 73,000 non-traumatic lower-limb amputations in 2010 were performed in adults aged 20 years or older with diagnosed diabetes, and approximately 60 percent of all non-traumatic lower-limb amputations occur in people with diabetes.

This paper appeared in: Behzad Gerami-Naini, et al., Cellular Reprogramming. June 2016, doi:10.1089/cell.2015.0087.

Encapsulation of Cardiac Stem Cells and Their Effect on the Heart


Earlier I blogged about an experiment that encapsulated mesenchymal stem cells into alginate hydrogels and implanted them into the hearts of rodents after a heart attack. The encapsulated mesenchymal stem cells showed much better retention in the heart and survival and elicited better healing and recovery of cardiac function than their non-encapsulated counterparts.

This idea seems to be catching on because another paper reports doing the same thing with cardiac stem cells extracted from heart biopsies. Audrey Mayfield and colleagues in the laboratory of Darryl Davis at the University of Ottawa Heart Institute and in collaboration with Duncan Steward and his colleagues from the Ottawa Hospital Research Institute used cardiac stem cells extracted from human patients that were encased in agarose hydrogels to treat mice that had suffered heart attacks. These experiments were reported in the journal Biomaterials (2013).

Cardiac stem cells (CSCs) were extracted from human patients who were already undergoing open heart procedures. Small biopsies were taken from the “atrial appendages” and cultured in cardiac explants medium for seven days.

atrial appendage

Migrating cells in the culture were harvested and encased in low melt agarose supplemented with human fibrinogen. To form a proper hydrogel, the cells/agarose mixture was added drop-wise to dimethylpolysiloxane (say that fast five times) and filtered. Filtration guaranteed that only small spheres (100 microns) were left. All the larger spheres were not used.

Those CSCs that were not encased in hydrogels were used for gene profiling studies. These studies showed that cultured CSCs expressed a series of cell adhesion molecules known as “integrins.” Integrins are 2-part proteins that are embedded in the cell membrane and consist of an “alpha” and “beta” subunit. Integrin subunits, however, come in many forms, and there are multiple alpha subunits and multiple beta subunits.

integrin-actin2

This mixing and matching of integrin subunits allows integrins to bind many different types of substrates. Consequently it is possible to know what kinds of molecules these cells will stick to based on the types of integrins they express. The gene prolifing experiments showed that CSC expressed integrin alpha-5 and the beta 1 and 3 subunits, which shows that CSC can adhere to fibronectin and fibrinogen.

fibronectin

fibrinogen-cleave

When encapsulated CSCs were supplemented with fibrinogen and fibronectin, CSCs showed better survival than their unencapsulated counterparts, and grew just as fast ans unencapsulated CSCs. Other experiments showed that the encapsulated CSCs made just as many healing molecules as the unencapsulated CSCs, and were able to attract circulating angiogenic (blood vessel making) cells. Also, the culture medium of the encapsulated cells was also just as potent as culture medium from suspended CSCs.

With these laboratory successes, encapsulated CSCs were used to treat non-obese diabetic mice with dysfunctional immune systems that had suffered a heart attack. The CSCs were injected into the heart, and some mice received encapsulated CSCs, other non-encapsulated CSCs, and others only buffer.

The encapsulated CSCs showed better retention in the heart; 2.5 times as many encapsulated CSCs were retained in the heart in comparison to the non-encapsulated CSCs. Also, the ejection fraction of the hearts that received the encapsulated CSCs increased from about 35% to almost 50%. Those hearts that had received the non-encapsulated CSCs showed an ejection fraction that increased from around 33% to about 39-40%. Those mice that had received buffer only showed deterioration of heart function (ejection fraction decreased from 36% to 28%). Also, the heart scar was much smaller in the hearts that had received encapsulated CSCs. Less than 10% of the heart tissue was scarred in those mice that received encapsulated CSCs, but 16% of the heart was scarred in the mice that received free CSCs. Those mice that received buffer had 20% of their hearts scarred.

Finally, did encapsulated CSCs engraft into the heart muscle? CSCs have been shown to differentiate into heart-specific tissues such as heart muscle, blood vessels, and heart connective tissue. Encapsulation might prevent CSCs from differentiating into heart-specific cell types and connecting to other heart tissues and integrating into the existing tissues. However, at this point, w have a problem with this paper. The text states that “encapsulated CSCs provided a two-fold increase in the number of engrafted human CSCs as compared transplant of non-encapsulated CSCs.” The problem is that the bar graft shown in the paper shows that the non-encapsulated CSCs have twice the engraftment of the capsulated CSCs. I think the reviewers might have missed this one. Nevertheless, the other data seem to show that encapsulation did not affect engraftment of the CSCs.

The conclusion of this paper is that “CSC capsulation provides an easy, fast and non-toxic way to treat the cells prior to injection through a clinically acceptable process.”

Hopefully large-animal tests will come next. If these are successful, then maybe human trials should be on the menu.