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.

Wound Healing Therapy That Combines Gene and Stem Cell Therapy


Researchers from Johns Hopkins University have examined wound healing in older mice and discovered that increasing blood flow to the wound can increase the rate of wound healing. Increasing blood flow to the wound requires a combination of gene therapy and the same stem cells the body already uses to heal itself.

John W. Harmon is professor of surgery at Johns Hopkins School of Medicine, and in a presentation to the American College of Surgeons’ Surgical Club, made the case that harnessing the power of bone marrow stem cells can increase the rate at which older people heal.

As we age, our wounds do not heal as fast. However, Harmon thinks that harnessing the power of bone marrow stem cells can remedy this disparity in healing rates.

To heal burns or other wounds, stem cells from the one marrow rush into action and home to the wound where they can differentiate into blood vessels, skin, and other reparative tissues. Stem cell homing is mediated by a protein called Hypoxia-Inducible-Factor-1 (HIF-1). According to Harmon, in older patients, few of these stem cells are released from the bone marrow and there is a deficiency of HIF-1. HIF-1 was actually discovered about 15 years ago by one of Harmon’s collaborators, a Johns Hopkins scientist named Gregg J. Semenza.

HIF-1
HIF-1

Harmon’s first strategy was to boost HIF-1 levels by means of gene therapy. This simply consisted of injecting the rodents with a copy of the HIF-1 gene that yielded higher levels of HIF-1 expression.

Even though higher levels of HIF-1 improved wound healing rates, burns were another story. To accelerate burn healing, Harmon and his co-workers used bone marrow stem cells from younger mice combined with the increased levels of HIF-1. This combination of HIF-1 and bone marrow stem cells from younger mice led to accelerated healing of burns so that after 17 days, almost all the mice had completely healed burns. These animals that healed so fast showed better blood flow to the wound and more blood vessels supplying the wound.

Harmon said that while this strategy is promising, he think that a procedure that uses a patient’s own bone marrow cells would work better since such cells would have a much lower chance of being rejected by the patient’s immune system. In the meantime, HIF-1 gene therapy has been successfully used in humans with a sudden lack of blood flow to a limb (see Rajagopalan S., et al., Circulation. 2007 Mar 13;115(10):1234-43). Harmon postulated that “it’s not a stretch of the imagination to think this could someday be used in elderly people with burns or other difficult wounds.”

A Step Towards Making Customized Blood Vessels


Johns Hopkins University scientists have directed stem cells to form networks of new blood vessels, and successfully transplanted those laboratory-made blood vessels into laboratory mice.

The stem cells in this experiment were made by reprogramming ordinary cells. Thus this new technique could potentially be used to make blood vessels that are genetically matched to individual patients and have a very low chance of being rejected by the patient’s immune system.

“In demonstrating the ability to rebuild a microvascular bed in a clinically relevant manner, we have made an important step toward the construction of blood vessels for therapeutic use,” said Sharon Gerecht, associate professor in the Johns Hopkins University Department of Chemical and Biomolecular Engineering, Physical Sciences-Oncology Center and Institute for NanoBioTechnology. “Our findings could yield more effective treatments for patients afflicted with burns, diabetic complications and other conditions in which vasculature function is compromised.”

Gerecht’s research group and others have previously grown blood vessels in the laboratory using stem cells, but there are problems with using these blood vessels in human patients. For example, in a paper published by Gerecht’s group in Stem Cells Translational Medicine earlier this year (Stem Cells Trans Med April 2013 vol. 2 no. 4 297-306), ECFCs or endothelial colony-forming cells from human umbilical cords were grown and used to make networks of blood vessels in culture. Those blood vessels were then embedded in blocks of “hyaluronic acid.” Hyaluronic acid is a component of human connective tissue, and when the ECFCs were embedded into it, they were then placed on the skin of mice that had received third-degree burns. On day 3 following transplantation, white blood cells called macrophages degraded the hyaluronic acid gel rather quickly. Between days 5–7, the mouse’s blood vessels infiltrated the implant and connected with the human blood vessels in the wound area. The growth of the human blood vessels peaked at day 7 and then decreased by the end of the proliferation stage. As the wound reached the remodeling period during the second week after transplantation, the blood vessels, including the transplanted human vessels generally regressed, and a few microvessels, wrapped by mouse smooth muscle cells and with a vessel area less than 200 square micrometers (including the human ones), remained in the healed wound.

This is a fascinating experiment, but making blood vessels this way is a heck of a lot of work, and even though the umbilical cord ECFCs are less likely to be rejected by the immune system of the patient, the chances of immune system rejection are still present. is there a better way?

In this current study, Gerecht and her team tried to streamline the new growth process. Where other experiments used chemical cues to differentiate stem cells into the desired cell type, Sravanti Kusuma in Gerecht’s laboratory devised a way to instruct stem cells to exclusively form the two cell types required for blood vessel construction (smooth muscle cells and endothelial cells).

According to Kusuma, “It makes the process quicker and more robust if you don’t have to sort through a lot of cells you don’t need to find the ones you do, or grow two batches of cells,”

Derivation of EVCs from hPSCs. (A) Schema for self-assembled vascular derivatives. (i) hPSCs are differentiated toward EVCs that can be matured into functional ECs and pericytes. (ii) Derived EVCs are embedded within a synthetic HA matrix that facilitates self-organization into vascular networks. (B) VEcad expression in day 12 differentiated hiPSC-MR31 and hESC-H9 cell lines comparing the three differentiation conditions tested (flow cytometry analysis; n = 3). (C and D) Flow cytometry plots (n = 3) of EVC derivatives assessing the expression of pluripotent markers TRA-1-60 and TRA-1-81 (C) and CD105 and CD146 (D). (E) EVC differentiation efficiency from hPSC lines per 1 million input hPSCs. (F) Flow cytometry plots (n = 3) of EVC derivatives assessing expression of VEcad double-labeled with CD105 or PDGFRβ. (Left) Isotype controls. (G) Quantitative RT-PCR analysis of EC and perivascular marker expression by EVCs and sorted VEcad+ and VEcad− cells. # denotes not detected. Data are normalized to EVCs of each specific hPSC type. (H) Flow cytometry plots (n = 3) of hematopoietic marker CD45 (hiPSC-BC1). (I) Quantitative RT-PCR of H9-EVCs for the expression of SMMHC and peripherin, compared with undifferentiated cells (d0) and mature derivatives (13, 37). Isotype controls for flow cytometry are in gray. Flow cytometry results shown are typical of the independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001
Derivation of EVCs from hPSCs. (A) Schema for self-assembled vascular derivatives. (i) hPSCs are differentiated toward EVCs that can be matured into functional ECs and pericytes. (ii) Derived EVCs are embedded within a synthetic HA matrix that facilitates self-organization into vascular networks. (B) VEcad expression in day 12 differentiated hiPSC-MR31 and hESC-H9 cell lines comparing the three differentiation conditions tested (flow cytometry analysis; n = 3). (C and D) Flow cytometry plots (n = 3) of EVC derivatives assessing the expression of pluripotent markers TRA-1-60 and TRA-1-81 (C) and CD105 and CD146 (D). (E) EVC differentiation efficiency from hPSC lines per 1 million input hPSCs. (F) Flow cytometry plots (n = 3) of EVC derivatives assessing expression of VEcad double-labeled with CD105 or PDGFRβ. (Left) Isotype controls. (G) Quantitative RT-PCR analysis of EC and perivascular marker expression by EVCs and sorted VEcad+ and VEcad− cells. # denotes not detected. Data are normalized to EVCs of each specific hPSC type. (H) Flow cytometry plots (n = 3) of hematopoietic marker CD45 (hiPSC-BC1). (I) Quantitative RT-PCR of H9-EVCs for the expression of SMMHC and peripherin, compared with undifferentiated cells (d0) and mature derivatives (13, 37). Isotype controls for flow cytometry are in gray. Flow cytometry results shown are typical of the independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001

Another difference from previous experiments was the use of induced pluripotent stem cells rather than bone marrow-derived endothelial precursor cells or umbilical cord-derived endothelial colony-forming cells. Gerecht’s team collaborated with Linzhao Cheng from the Institute for Cell Engineering to co-opt her expertise with induced pluripotent stem cells (iPSCs), which are made from adult cells and are de-differentiated through genetic engineering techniques to become embryonic-like stem cells.

Cheng said that this experiment is “an elegant use of human induced pluripotent stem cells that can form multiple cell types within one kind of tissue or organ and have the same genetic background [as the patient].” Cheng continued” “In addition to being able to form blood cells and neural cells as previously shown, blood-derived human induced pluripotent stem cells can also form multiple types of vascular network cells.”

To grow blood vessels, Cheng, Gerecht and others placed the stem cells into a scaffolding made of hydrogel (hyaluronic acid and water). This hydrogel was full of chemical cues that directed the cells to differentiate in to endothelial and smooth muscle cells and form a network of blood vessels. This constitutes the first time human blood vessels had been made from human pluripotent stem cells in a synthetic material.

Self-assembly of EVCs to multicellular networks in a 3D matrix. (A) Network formation from BC1-EVCs in collagen (i) and HA hydrogels (ii). (B) Sorted VEcad+ and VEcad− cells encapsulated within collagen gels were unable to form networks. (Insert) Example of a cell with typical stellate morphology, with phalloidin in green and nuclei in blue. (Scale bars: 100 μm.) (C) Vacuole formation was observed after one day as evidenced by light microscopy (LM) (i) and confocal images (ii) of vacuole vital stain, FM4-64, in red and nuclei in blue. (Scale bar: 10 μm.) (D) On day 2, network formation with enlarged lumen (i and ii) and cell sprouting (iii and iv) were visualized by LM images (i and iii) and confocal images (ii and iv) of FM4-64 in red and nuclei in blue. (Scale bars: 10 μm in i and iii; 20 μm in ii; 50 μm in iv.) (E) On day 3, complex networks were observed with enlarged and open lumen, as indicated by confocal z-stacks and orthogonal sections of FM4-64 in red and nuclei in blue. (Scale bar: 20 μm.) (F) After 3 d, multilayered structures were also detected, as demonstrated by a 3D projection image of NG2 (green), phalloidin (red), and nuclei (blue) showing NG2+ pericytes integrated onto hollow structures. Images shown are typical of the independent experiment. (Scale bars: 50 µm.)
Self-assembly of EVCs to multicellular networks in a 3D matrix. (A) Network formation from BC1-EVCs in collagen (i) and HA hydrogels (ii). (B) Sorted VEcad+ and VEcad− cells encapsulated within collagen gels were unable to form networks. (Insert) Example of a cell with typical stellate morphology, with phalloidin in green and nuclei in blue. (Scale bars: 100 μm.) (C) Vacuole formation was observed after one day as evidenced by light microscopy (LM) (i) and confocal images (ii) of vacuole vital stain, FM4-64, in red and nuclei in blue. (Scale bar: 10 μm.) (D) On day 2, network formation with enlarged lumen (i and ii) and cell sprouting (iii and iv) were visualized by LM images (i and iii) and confocal images (ii and iv) of FM4-64 in red and nuclei in blue. (Scale bars: 10 μm in i and iii; 20 μm in ii; 50 μm in iv.) (E) On day 3, complex networks were observed with enlarged and open lumen, as indicated by confocal z-stacks and orthogonal sections of FM4-64 in red and nuclei in blue. (Scale bar: 20 μm.) (F) After 3 d, multilayered structures were also detected, as demonstrated by a 3D projection image of NG2 (green), phalloidin (red), and nuclei (blue) showing NG2+ pericytes integrated onto hollow structures. Images shown are typical of the independent experiment. (Scale bars: 50 µm.)

While these networks of blood vessels looked like the real thing, would they work within a living creature? The answer that question, Gerecht and her group transplanted them into mice. After two weeks the lab-grown blood vessels had integrated with the mouse’s own blood vessels and the hydrogel had dissolved and been degraded. “That these vessels survive and function inside a living animal is a crucial step in getting them to medical application,” Kusama said.

An important follow-up to these experiments is to examine the three-dimensional structure of these blood vessels to determine if truly have all the characteristics of human blood vessels that can deliver blood to damaged tissues and help those tissues recover from injury or trauma.