Bone-Making Ability of Amniotic Epithelial Cells


Human amniotic epithelial cells (hAECs) develop from the embryo as early as eight days after fertilization. hAECs have been shown to possess remarkable plasticity, or the ability to form many cell types. Mattioli and others showed that AECs from sheep have capacity to differentiate into bone cells (see Cell Biol Int 2012;36:7-19). Therefore, it is possible that these cells could be developed into a source of cells for regenerative treatments.

Steve Shen from the Shanghai Jiao Tong University School of Medicine and his coworkers have examined the ability of cultured hAECs to make bone in culture, and then applied their techniques to an animal system to test the ability of cultured hAECs to restore tooth sockets and facial bone.

To begin, hAECs were isolated from healthy mothers who had undergone childbirth by cesarean section. These cells were cultured in a standard cell culture medium (DMEM/F12 for those who are interested), expanded in culture, and then subjected to flow cytometry to isolate those cells with all the right cell surface markers.

These cells wee then placed into a specialized culture medium designed to convert mesenchymal stem cells into bone-making cells. This osteogenic induction medium was changed every three days for 21 days.  The figure below shows how well the bone-induction worked.  A red stain called alizarin red binds to calcium and therefore stains forming bone matrices rather well.  As you can see in the panel labeled “G” below, the hAECs grown in the osteogenic medium stain red rather well, which shows that they are making bone.

Characterization of hAECs in vitro. (A, B): hAECs at passages 0 and 1 displayed a cobblestone-like morphology. (C): Some hAECs changed into fibroblast-like cells after 7 days of osteoblastic culture. (D): hAECs showed significant morphological changes and settled on superimposed layers after 21 days of osteoblastic culture. (E): Cell proliferation of hAECs at passage 1 was significantly higher than at passages 0 and 5 from day 4 to day 12; hAECs at passage 5 displayed the lowest proliferation rate from day 6 to day 14 (☆, p < .05). (F): Flow cytometry analysis showed that hAECs expressed CD44, CD90, CD105, and SSEA-4 and did not express CD34, CD45, and HLA-DR. Values represent the percentages of all assessed cells positively stained by the indicated antigens (bottom of each graph). Nonspecific fluorescence was determined as the blank control using isotype-matched monoclonal antibodies (PE blank, FITC blank). (G): Representative images of microscopic and general photographs for ALP and ARS staining in osteogenic and control groups indicated the osteogenic differentiation of hAECs in vitro. Scale bar = 200 μm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; FITC, fluorescein isothiocyanate; hAEC, human amniotic epithelial cell; OD, optical density; p, passage; PE, phycoerythrin.
Characterization of hAECs in vitro. (A, B): hAECs at passages 0 and 1 displayed a cobblestone-like morphology. (C): Some hAECs changed into fibroblast-like cells after 7 days of osteoblastic culture. (D): hAECs showed significant morphological changes and settled on superimposed layers after 21 days of osteoblastic culture. (E): Cell proliferation of hAECs at passage 1 was significantly higher than at passages 0 and 5 from day 4 to day 12; hAECs at passage 5 displayed the lowest proliferation rate from day 6 to day 14 (☆, p < .05). (F): Flow cytometry analysis showed that hAECs expressed CD44, CD90, CD105, and SSEA-4 and did not express CD34, CD45, and HLA-DR. Values represent the percentages of all assessed cells positively stained by the indicated antigens (bottom of each graph). Nonspecific fluorescence was determined as the blank control using isotype-matched monoclonal antibodies (PE blank, FITC blank). (G): Representative images of microscopic and general photographs for ALP and ARS staining in osteogenic and control groups indicated the osteogenic differentiation of hAECs in vitro. Scale bar = 200 μm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; FITC, fluorescein isothiocyanate; hAEC, human amniotic epithelial cell; OD, optical density; p, passage; PE, phycoerythrin.

When hAECs were subjected to gene expression assays, it was clear that the cells grown in the osteogenic medium expressed a whole host of bone-specific genes.  Therefore, the formation of bone was not a fluke, since these cells not only concentrated calcium, but also increased their expression of bone-specific genes (these include osterix, Runx2, Alkaline phosphatase, Collagen I, and osteoprotegerin).

Additionally, those hAECs grown in osteogenic medium stopped forming sheets of cells and began to grow as individual cells.  This is an important transformation for the synthesis of bone because bone-making cells secrete a bone-specific protein matrix upon which calcium phosphate-based crystals deposit to form bone.  If the cells were organized in sheets, then bone deposition would not be possible.  When isolated from amniotic membranes, hAECs grow as sheets of cells that are known as epithelia.  In order to become bone-making cells, the hAECs must undergo an “epithelial-to-mesenchymal transformation” or EMT.  This requires turning on new genes and turning off others.  Gene expression assays of hAECs grown in the osteogenic medium, show extensive evidence of EMT.  For example, a protein called E-cadherin is essential for cells growing in sheets, because it helps cells stick to each other.  However, in hAECs grown in the osteogenic medium, E-cadherin expression was quite low.  Also, a protein called vimentin is highly expressed during EMT, and the hAECs grown in osteogenic medium showed high expression of vimentin.  Thus, these hAECs were undergoing all the necessary changes in order to become bone-making cells,, making all the right genes, and made bone in culture to boot.

This is certainly interesting, but can these hAECs repair bone in a living animal?  Shen’s group tried that very experiment.  The hAECs that were grown in the osteogenic medium were loaded on tricalcium phosphate scaffolds and implanted into rodents with tooth socket lesions.  Control animals were implanted with tricalcium phosphate scaffolds without cells.  The scaffold with cells significantly increased bone formation in the rodents, and showed much more infilling with mineralized tissue.  There was also extensive evidence of the formation of new vasculature and wandering cells called macrophages that are important for the degradation of the implanted scaffold required for new bone formation.  Tissue samples were examined 4 to 8 weeks after the implants were placed.

In vivo healing process in alveolar defect at 4 and 8 weeks postoperatively. (A): Representative three-dimensional micro-computed tomography (CT) reconstruction images of hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 1 mm. (B): Micro-CT parameters acquired among β-TCP scaffold in vitro, hAECs+β-TCP scaffold in vivo, and β-TCP scaffold alone in vivo at 4 and 8 weeks postoperatively (☆, p < .05). (C): Hematoxylin and eosin staining of the rat alveolar defect at 4 and 8 weeks postoperatively revealed more active new bone formation in the EXP group than in the CTR group (×50 and ×200 magnification). Alveolar defect treated with hAECs+β-TCP scaffold exhibited a more mature lamellae-bone formation at 8 weeks postoperatively. Scale bar = 200 μm. (D): ANA-positive cells, visible as green fluorescence in the nuclei, were observed within the newly deposited OCN, and OPN-positive bone tissue, visible as red fluorescence, in the EXP group at 4 weeks postoperatively, indicating a mature osteoblastic function of these hAEC-derived cells. Scale bar = 50 μm. (E): Representative images of immunohistochemical staining of sections with anti-VEGF antibody and anti-CD68 antibody in hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 200 μm. (F): The histomorphometric quantification of the relative new bone area, VEGF-positive area, and CD68-positive area showed that more bone tissue regeneration was observed in the EXP group than in the CTR group at 4 and 8 weeks postoperatively. The positive signal of VEGF and CD68 in the EXP group was much weaker at 4 weeks postoperatively and became more intense at 8 weeks postoperatively compared with the CTR group (☆, p < .05). Abbreviations: ANA, anti-nuclear antibody; BV/TV, bone volume/tissue volume ratio; BMD, bone mineralization density; hAECs, human amniotic epithelial cells; OPN, osteopontin; post op, postoperatively; SMI, structure model index; Tb.Th., trabecular thickness; Tb.N., trabecular number; Tb.Sp., trabecular separation; β-TCP, β-tricalcium phosphate; VEGF, vascular endothelial growth factor.
In vivo healing process in alveolar defect at 4 and 8 weeks postoperatively. (A): Representative three-dimensional micro-computed tomography (CT) reconstruction images of hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 1 mm. (B): Micro-CT parameters acquired among β-TCP scaffold in vitro, hAECs+β-TCP scaffold in vivo, and β-TCP scaffold alone in vivo at 4 and 8 weeks postoperatively (☆, p < .05). (C): Hematoxylin and eosin staining of the rat alveolar defect at 4 and 8 weeks postoperatively revealed more active new bone formation in the EXP group than in the CTR group (×50 and ×200 magnification). Alveolar defect treated with hAECs+β-TCP scaffold exhibited a more mature lamellae-bone formation at 8 weeks postoperatively. Scale bar = 200 μm. (D): ANA-positive cells, visible as green fluorescence in the nuclei, were observed within the newly deposited OCN, and OPN-positive bone tissue, visible as red fluorescence, in the EXP group at 4 weeks postoperatively, indicating a mature osteoblastic function of these hAEC-derived cells. Scale bar = 50 μm. (E): Representative images of immunohistochemical staining of sections with anti-VEGF antibody and anti-CD68 antibody in hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 200 μm. (F): The histomorphometric quantification of the relative new bone area, VEGF-positive area, and CD68-positive area showed that more bone tissue regeneration was observed in the EXP group than in the CTR group at 4 and 8 weeks postoperatively. The positive signal of VEGF and CD68 in the EXP group was much weaker at 4 weeks postoperatively and became more intense at 8 weeks postoperatively compared with the CTR group (☆, p < .05). Abbreviations: ANA, anti-nuclear antibody; BV/TV, bone volume/tissue volume ratio; BMD, bone mineralization density; hAECs, human amniotic epithelial cells; OPN, osteopontin; post op, postoperatively; SMI, structure model index; Tb.Th., trabecular thickness; Tb.N., trabecular number; Tb.Sp., trabecular separation; β-TCP, β-tricalcium phosphate; VEGF, vascular endothelial growth factor.

hAECs are typically not recognized by the immune system as foreign and they also have anti-scarring capabilities.  Not only can they effectively form bone in culture, but they can also repair an alveolar defect in a rodent model.  Clearly these cells show promise for clinical applications.  However, before they can be used in the clinic more has to be known about their culture conditions, how many cells are required for transplantation, the best cell dose, at what passages they need to be used, and so on.  Thus, this paper represents a good start for hAECs, but they have to be better understood before they can come to the clinic.

Direct Reprogramming Cells with Recombinant Proteins


In my opinion, for what it’s worth, we will probably see direct reprogramming take a prominent place in regenerative medicine in the future. It will not be in the near future, but as direct reprogramming becomes better understood and more feasible, it will probably become a central part of the discussion of regenerative medical strategies.

Direct reprogramming, which is also known as lineage conversion, uses cell type-specific transcription factors to convert a mature, adult cell into a different type of mature, adult cell. The cell does not pass through a pluripotent intermediate, and becomes a wholly different type of cell.

Of course, forcing the expression of lineage-specific transcription factors in cells requires that they be treated with recombinant viruses or other such tools. These genetic manipulations present problems for regenerative medicine, since such viruses can cause mutations or cause the introduced genes to be constantly activated, both of which can cause cells to die to grow uncontrollably. Genetically engineering cells needs to be done in a “kinder and gentler” way (to quote George HW Bush).

To that end Dennis Clegg and his colleagues from the Center for Stem Cell Biology and Engineering at UC Santa Barbara have used specially designed proteins to directly cultured retinal pigmented epithelial cells to neurons.

Newly discovered C-end rule (CendR) cell- and tissue-penetrating peptides have a arginine-rich sequence at the end of proteins that allows them to bind particular cell receptors and be internalized into the cell. These CendR peptides bind to the NRP-1 protein and are internalized. Several laboratories have used CendR peptides to increase the efficacy of anti-cancer drugs in experimental cases (see Alberici L, et al (2013) Cancer Res 73:804–812; Sugahara KN, et al. (2010) Science 328:1031–1035; Sugahara KN, et al. (2009) Cancer Cell 16:510–520; and Roth L,, et al. (2012) Oncogene 31:3754–3763).

By tacking a CendR peptide to the end of the Sox2 protein, Clegg and others were able to convert retinal pigmented epithelial (RPEs) cells to neurons. The Sox2 protein is highly expressed in neural progenitor cells. Other studies have shown that Sox2 can reprogram mouse and human fibroblasts to neural stem cells (Ring KL, et al. (2012) Cell Stem Cell 11:100–109). Thus, Sox2 should do the trick.

Making cultured RPE cells from embryonic stem cells is relatively easy to do. Therefore, Clegg and his coworkers made cultured RPEs and then treated them with viruses that expressed Sox2. The cultured RPEs showed conversion to neurons and the expression of neuron-specific genes.

Since they had established that Sox2 could convert RPEs to neurons, they tried recombinant Sox2 protein with the CendR peptide RPARPAR at the end of the protein. After 60 days in culture, the cells expressed a host of neuron-specific genes, and were capable of taking up a dye that only active neurons can take up (FM1-43).

Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.
Reprogramming human fetal RPE (hfRPE) cells to neurons using recombinant SOX2 proteins. (A): Efficiency of hfRPE cells to be reprogrammed to neuron-like cells after recombinant proteins was added to the media every 24 hours for 30 days. (B): Efficiency of hfRPE cells to be reprogrammed by adding SOX2-RPARPAR recombinant protein every 48 hours for different time courses. (C): Representative images of hfRPE (fRPE1914) cells during reprogramming to neuron-like cells after 30, 40, and 50 days in culture with SOX2-RPARPAR protein. Scale bars = 100 μm. (D): Representative images of hfRPE (fRPE1914) cells reprogrammed to neuron-like cells expressing neuronal markers, but not an RPE marker (PAX6), using SOX2-RPARPAR protein. Scale bars = 50 μm. Abbreviations: D, days; RPE, retinal pigmented epithelial cells.

The efficiency for this experiment was lousy (0.3%) as opposed to the efficiency for the use of recombinant viruses (11%). Nevertheless, this experiment shows that it is possible to directly reprogram cells without using recombinant viruses.

2014 in review


The WordPress.com stats helper monkeys prepared a 2014 annual report for this blog.

Here’s an excerpt:

Madison Square Garden can seat 20,000 people for a concert. This blog was viewed about 67,000 times in 2014. If it were a concert at Madison Square Garden, it would take about 3 sold-out performances for that many people to see it.

Click here to see the complete report.

A New Way to Extract Bone-Making Cells From Fat


With the holiday season upon us, many of us tend to put on a few extra pounds of fat. However, fat also is a reservoir of mesenchymal stem cells (MSCs) that can make cartilage, bone or even more fat.

A new study by scientists at Brown University demonstrate the efficacy of a new method for extracting potential bone-making cells from human fat. To perfect their technique, the Brown University team, led by senior author Eric Darling, developed a fluorescent tag that identified any cell that expressed the ALPL (alkaline phosphatase liver/bone/kidney) gene. This probe is a fluorescent, oligodeoxynucleotide molecular beacon probe specific for ALPL mRNA.  This probe hybridizes to the ALPL mRNA and fluoresces strongly as a result of the hydridization.  Fluorescence-activated cell sorting ten isolates the ALPL-expressing cells from the non-expression cells effectively and easily.

Molecular beacon probes are single-stranded nucleic acid molecules that have a fluorescent dye attached to one end and a quenching molecule that prevents the dye from fluorescing at the other end.  A short, complementary sequence at either end of the probe causes the probe to self-hybridize, thus bringing the glowing molecule and its inhibitor close together.  Under these conditions, the probe does not glow.  However, if the probe hybridizes to a specific sequence, then the glowing molecule and its inhibitor are far apart and the glowing molecule fluoresces.

Molecular Beacons hybridize to their specific target sequence causing the hairpin-loop structure to open and separate the 5’ end reporter from the 3’ end quencher. As the quencher is no longer in proximity to the reporter, fluorescence emission takes place. The measured fluorescence signal is directly proportional to the amount of target DNA.
Molecular Beacons hybridize to their specific target sequence causing the hairpin-loop structure to open and separate the 5’ end reporter from the 3’ end quencher. As the quencher is no longer in proximity to the reporter, fluorescence emission takes place. The measured fluorescence signal is directly proportional to the amount of target DNA.

The level of ALPL gene expression strongly correlates with the ability of cells to form bone. According to Darling and his team, their technique more than doubled the quantity of bone-making cells that could be extracted from fat.  Brown University has applied for a patent on this method.

In culture, ALPL-expressing cells produced, on average, twice as much bony matrix as other cells. ALPL-expressing cells also were better at making cartilage or even fat. Even though other groups have isolated cells on the basis of gene expression, none of the available published techniques, to date, have used gene expression-based isolation to enrich for cell populations of a specific tissue, such as bone.

The lead author of this work, Hetal Marble, said that targeting gene expression rather than cell surface proteins is a relatively new paradigm for cell isolation and purification. Because using cell surface proteins always requires accepting certain assumptions about the cell types that are being isolated, gene expression-based isolation is a more pragmatic way to approach the problem. Consider this: cells that are in certain tissues express certain genes characteristic of that tissue. Therefore, using those gene expression patterns to isolate such cells leaves doubt that the cells you will isolate are in the process of differentiating into the desired tissue. Using cell surface proteins, however, is less precise because particular cell populations rarely are the only cell types that express that protein.

“Approaches like this allow us to isolate all the cells that are capable of doing what we want, whether they fit the archetype of what a stem cell is or not,” said Marble. “The paradigm shift is thinking about populations that are able to achieve am end point rather than isolating populations that fit a strictly defined archetype.”

In this experiment, Darling’s group used a special culture medium designed to crank up bone-specific gene expression in sensitive cells. This so-called “priming step” took four days in culture before the ALPL gene expression levels were high enough to properly detect it. However, in the future, Darling believes that by targeting a gene whose levels of expression increase earlier in during the process of differentiation, he and his team should be able to dispense with the four-day delay. This way, their technique would be applicable in the operating room, since surgeons could isolate fat from the patient with a bone break and prime it (or not), and then isolate the bone-making cells from the fat, which they would use to treat their patient’ fracture.

“If you can take the patient into the OR, isolate a bunch of their cells, sort them, and put them back in that’s ideally where we’d like to go with this,” said Darling. “Theoretically we could do this with other genes that might upregulate very quickly or are innately expressed.”

A “SMARTer” Way to Isolate Mesenchymal Stem Cells


The Singapore-MIT Alliance for Research and Technology or SMART employs a team of engineers and life scientists to design technologies that address problems in science and medicine. In particular, a SMART team has devised a new technique to identify mesenchymal stem cells from bone marrow cells on the basis of cell size, cell stiffness, and the deformation of the nucleus.

Mesenchymal stem cells (MSCs) constitute less than one percent of the total cells in bone marrow. Therefore, isolating these cells from the morass of cells that are in the bone marrow is somewhat of a challenge. Most of the procedures for isolating MSCs from bone marrow utilize cell surface proteins found on the surfaces of MSCs, but there are no few cell surface proteins that are unique to only MSCs. Therefore, such isolation procedures tend to be tedious and not completely efficient. Because MSCs can differentiate into cells that produce bone, cartilage, fat, or muscle, they have proven invaluable for tissue repair therapies.

This new study by the SMART team has identified three physical characteristics of MSCs that can distinguish them from other immature cells found in the bone marrow. These physical characteristics should help them invent devices that could rapidly isolate MSCs, and facilitate the isolation of sufficient numbers of stem cells to treat patients.

Presently there are no sure-fire ways to quickly and efficiently separate MSCs from bone marrow cells that have already begun to differentiate into other cell types, but share the same molecules on the cell surface. This caveat may explain why experimental results vary among labs, and why stem-cell treatments now in clinical trials are not as effective as they could be, said Krystyn Van Vliet, an MIT associate professor of materials science and engineering and biological engineering and a senior author of the paper, that appeared in the Proceedings of the National Academy of Sciences.

“Some of the cells that you’re putting in and calling stem cells are producing a beneficial therapeutic outcome, but many of the cells that you’re putting in are not,” Van Vliet said. “Our approach provides a way to purify or highly enrich for the stem cells in that population. You can now find the needles in the haystack and use them for human therapy.”

In bone marrow, MSCs exist alongside other immature cells, such as osteogenic cells, which have already begun the developmental path toward becoming cartilage- or bone-producing cells. Currently, researchers try to isolate MSCs based on protein markers found on the cell surfaces, but these markers are not specific to MSCs. Therefore isolation techniques that rely on cell surface proteins can also co-isolate other types of immature cells that are more differentiated.

“Conventional cell-surface markers are frequently used to isolate different types of stem cells from the human bone marrow, but they lack sufficient ‘resolution’ to distinguish between subpopulations of mesenchymal stromal cells with distinct functions,” Lee said.

The researchers set out to find biophysical markers for multipotency (the ability to differentiate into several different cell types). They hypothesized that cell size might be a factor, since fetal bone marrow stem cells, which tend to have a higher percentage of MSCs, are usually small in diameter.

Jongyoon Han, an MIT professor of electrical engineering and biological engineering, had previously invented a device that captures circulating tumor cells based on their size. The SMART team used Han’s machine to isolate bone marrow cells based on size and discovered that none of the larger cells were multipotent, but not all of the smaller cells were multipotent. Therefore, size alone in insufficient to distinguish MSCs.

After measuring several other physical traits, the SMART team observed that two other physical characteristics could be combined with cell size to completely distinguish MSCs from other stem cells: stiffness of the cell, and the degree of fluctuation in the cell’s nuclear membrane.

“You don’t need more than these three, but you also can’t use fewer than these three,” Van Vliet said. “We now have a triplet of characteristics that identifies populations of cells that are going to be multipotent versus populations of cells that are only going to be able to become bone or cartilage cells.”

These features seem to correspond to what is already known about stem cells, Van Vliet said. In contrast to cells that have already committed to their final fate, immature cells have genetic material that moves around inside the nucleus, producing more fluctuations of the nuclear cell membrane. Stem cells also have a less rigid internal cytoskeletal structure than those of highly differentiated cells, which makes them seem less stiff.

The researchers then tested the regenerative abilities of MSCs isolated on the basis of these three characteristics in mice. They found that immature MSCs could help repair both muscle and bone injuries, but cells identified as osteogenic stromal cells were able to repair bone but not muscle.

“We have provided the first demonstration that subpopulations of mesenchymal stromal cells can be identified and highly enriched for bone growth and muscle repair,” Lee said. “We envision that this approach would also be important in the selection and purification of bone marrow-derived stem cells for tissue repair in human patients suffering from a range of tissue-degenerative diseases.”

“This is potentially a big step forward in establishing a marker-free way of identifying mesenchymal stem cells with maximum differentiation capacity,” said Jochen Guck, a professor of cellular machines at the Dresden University of Technology. “Biophysical markers have long been discussed and sought as an alternative to antibody labeling. What sets this work apart from others is that it clearly said that no single marker (at least of those tried) alone is predictive enough, but that a combination of them is required.”

The SMART team is now working on high-speed methods for separating MSCs. Creating more pure populations of such cells should lead to more effective stem-cell treatments for tissue injuries, Van Vliet said.

“Instead of putting in 30 percent of the cells that you want, and 70 percent filler, you’re putting in 100 percent of the cells that you want,” she explains. “That should lead to more reliable patient outcomes, because you’re not going to have this variability from batch to batch, or patient to patient, in how many of each cell population are present.”

Van Vliet and Poon also hope to initiate a clinical trial that utilizes the osteogenic cells isolated in this study, which could potentially prove useful for treating bone injuries.

Bone Marrow Stem Cells and Tissue Engineering Give a Woman a New Smile


Massive injuries to the face can cause bone loss and “tooth avulsion.” Medically speaking, avulsion simply refers to the detachment of a body structure from its normal location by means of surgery or trauma. Dental implants and help with lost teeth, but if the facial bone has suffered so much loss that you cannot place implants in them, then you are out of luck. Dental prostheses can help, but these do not always fit very well.

Darnell Kaigler and his group at the University of Michigan Center for Oral Health wanted to help a 45-year-old woman who had lost seven teeth and a good portion of her upper jaw bone (maxilla) as a result of massive trauma to the face. This poor lady had some dentures that did not fit well and a mouth that did not work well.

Bone can be grown from stem cells, but getting those stem cells to survive and do what you want them to do is the challenge of regenerative medicine. Therefore, Dr. Kraigler and his group used a new technique to help this young lady, and their results are reported in the December 2014 issue of the journal Stem Cells Translational Medicine.

First, Kraigler and his co-workers extracted bone marrow stem cells from a bone marrow aspiration that was taken from the upper part of the hip bone (the posterior crest of the ilium for those who are interested).  They used a product called ixmyelocel-T from Aastrom Biosciences in Ann Arbor , MI. This product is a patient-specific, expanded multicellular therapy, cell-processing system that selectively expands mesenchymal cells, monocytes and alternatively activated macrophages, up to several hundred times more than the number found in the patient’s bone marrow, while retaining many of the hematopoietic cells collected from only a small sample (50ml) of the patient’s bone marrow. Thus, the healing cells from the bone marrow are grown and made healthy, after which the cells were bagged and frozen for later use.

ixmyelocel-T

Then the patient was readied for the procedure by having the gum tissue cut and lifted as a flap of tissue (under anesthesia, or course). Then four holes were drilled into the bone and setting screws were inserted. This is an important procedure, because implanted stem cells will not survive unless they have blood vessels that can bring them oxygen and nutrients. By drilling these holes, the tissue responds by making new blood vessels. To this exposed surface, the bone marrow-derived stem cells were applied with a tricalcium phosphate (TCP). TCP is a salt that will induce mesenchymal stem cells to form bone. Once the TCP + stem cell mixture was applied to the gum, a collagen membrane was placed over it, and the gum was then sewn shut with sutures.

Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.
Cell transplantation procedure. Front view (A) and top view (B) of the initial clinical presentation showing severe hard and soft tissue alveolar ridge defects of the upper jaw. Following elevation of a full-thickness gingival flap, the images show front view (C) and top view (D) of the severely deficient alveolar ridge, clinically measuring a width of only 2–4 mm. Front view (E) and top view (F) of the placement of “tenting” screws in preparation of the bony site to receive the graft. Placement of the β-tricalcium phosphate (seeded with the cells 30 minutes prior to placement at room temperature) into the defect (G), with additional application of the cell suspension following placement of the graft in the recipient site (H). Placement of a resorbable barrier membrane (I) to stabilize and contain the graft within the recipient site, and top view (J) of primary closure of the flap.

Four months later, the patient underwent a cone-beam computed tomography (CBCT) scan. The bone regrowth can be seen in the figure below.

Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.
Cone-beam computed tomography (CBCT) scans. CBCT scans were used to render three-dimensional reconstructions of the anterior segment of the upper jaw and cross-sectional (top view) radiographic images to show volumetric changes of the upper jaw at three time points. (A, B): The initial clinical presentation shows 75% jawbone width deficiency. (C, D): Immediately following cell therapy grafting, there is full restoration of jawbone width. (E, F): Images show 25% resorption of graft at 4 months and overall net 80% regeneration of the original ridge-width deficiency.

According to the paper, there was an “80% regeneration of the original jawbone.”

Into this newly regenerated bone, permanent dental implants were placed. The results are shown below.

Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.
Complete oral rehabilitation. Clinical presentation of the patient prior to initiation of treatment (A) and following completed oral reconstruction (B). (C): Periapical radiographs of oral implants showing osseointegration of implants and stable bone levels at the time of placement, 6 months following placement, and 6 months following functional restoration and biomechanical loading of implants with a dental prosthesis.

Pardon me, but permit me an unprofessional moment when I say that this is really cool.  Of course, this patient will need to be observed over the next several years to determine the longevity of her bone regeneration, but the initial result is certainly something to be excited about.

Tricalcium phosphate or TCP has been used to induce the bone-making activities of mesenchymal stem cells.  It has also been used in several animal studies as a delivery vehicle for mesenchymal stem cells (for example, see Rai B, et al., Biomaterials 2010, 31:79607970; Krebsbach PH, et al., Transplantation 1997, 63:10591069; Zhou J, et al., Biomaterials 2010, 31:11711179).  TCP also seems to support stem cell proliferation, survival, and differentiation into bone.  Kresbach and others showed that TCP most consistently yielded bone formation when used as a delivery vehicle for mesenchymal stem cells compared to other biomaterials commonly used, such as gelatin sponges and demineralized bone matrix.  However, there are no studies that have ascertained how well stem cells attach to TCP, and this attachment is an important factor in determining how many stem cells reach the site of injury.  This study by Kaigler and his group (A. Rajan and others) showed that a 30-minute incubation of the cells with TCP gave sufficient attachment of the cells to the TCP for clinical use.  The efficiency of this incubation period was also not affected by the temperature.  

The other exciting features of this paper, is that most of the materials used in this study were commercially available.  The bone marrow stem cell isolation technique was pioneered by Dennis JE, and others in their 2007 article in the journal Stem Cells (25:25752582).  Effective commercialization of this technique has shown the efficacy of this procedure for clinical use.  This paper also shows the clinical feasibility of using TCP as a delivery vehicle for mesenchymal stem cell-based bone treatments.

In conclusion, I will quote the authors: “Cell survival and seeding efficiency in the context of tissue engineering and cell-therapy strategies are critical parameters for success that have not been rigorously examined in a clinical context. This study defined optimized conditions for these parameters using an autologous stem cell therapy to successfully treat a patient who had a debilitating craniofacial traumatic deficiency. To our knowledge, there have been no other clinical reports of cell therapy for the treatment of craniofacial trauma defects. This clinical report serves as solid foundation on which to develop more expanded studies using this approach for the treatment of larger numbers of patients with other debilitating conditions (e.g., congenital disorders) to further evaluate efficacy and feasibility.”

Telling a Good Batch of Mesenchymal Stem Cells from a Lousy One


If a clinician isolates mesenchymal stem cells from the fat, bone marrow, or muscles of a patient, how can they tell if these cells will be effective? Short answer – they can’t. How well the cells grow in culture and how they look is the best indicator to date, but these indicators can fool you.

Fortunately, the highly productive laboratory of Darwin Prockop from the Institute of Regenerative Medicine at Texas A & M has discovered that the expression of a gene called TSG-6 can act as an indicator for human bone marrow mesenchymal stem cell quality.

In this paper, Prockop and others examined mice that had suffered damage to the surfaces of their eyes (corneas). To mitigate the inflammation in the eye, Prockop and his colleagues applied bone marrow-derived mesenchymal stem cells, but it was clear that the stem cell batches varied remarkably in their ability to regulate inflammation.

Prockop and his group then examined the genes expressed in the different batches of bone marrow-derived mesenchymal stem cells (MSCs) in order to determine if there was a gene that was consistently expressed in the effective batches as opposed to the ineffective batches.

Fortunately, they hit pay dirt. Reverse Transcriptase-PCR assays of human MSCs for the TSG-6 gene accurately predicted their ability of a specific batch of cells to modulate inflammation during corneal injury, or damage to the body wall (sterile peritonitis), or drug-induced injury to the lung. Thus, if you want implanted MSCs to modulate inflammation, then you want cells that express TSG-6 at a high level.

However, if you want MSCs that make bone, then you do not want cells that express high levels of TSG-6 because the levels of TSG-6 mRNA were negatively correlated with their potential for osteogenic (bone cell) differentiation in culture.

Additionally, when donated MSCs from male and female donors were examined and compared, it was clear that MSCs from female donors more effectively suppressed sterile inflammation, expressed higher levels of TSG-6, and had slightly less osteogenic potential than those from male donors.

Thus, TSG-6 is a marker that can measure the ability of a batch of MSCs to suppress inflammation. It is unclear if this same gene marker equally applies to other types of MSCs, but that will hopefully become clear with further work. Also, markers that correlate with the ability of these cells to do other types of regenerative activities might also result from experiments like these.

Merry Christmas to All My Readers


Matthew 1:18-25:

18 This is how the birth of Jesus the Messiah came about: His mother Mary was pledged to be married to Joseph, but before they came together, she was found to be pregnant through the Holy Spirit. 19 Because Joseph her husband was faithful to the law, and yet did not want to expose her to public disgrace, he had in mind to divorce her quietly.

20 But after he had considered this, an angel of the Lord appeared to him in a dream and said, “Joseph son of David, do not be afraid to take Mary home as your wife, because what is conceived in her is from the Holy Spirit. 21 She will give birth to a son, and you are to give him the name Jesus, because he will save his people from their sins.”

22 All this took place to fulfill what the Lord had said through the prophet: 23 “The virgin will conceive and give birth to a son, and they will call him Immanuel” (which means “God with us”).

24 When Joseph woke up, he did what the angel of the Lord had commanded him and took Mary home as his wife. 25 But he did not consummate their marriage until she gave birth to a son. And he gave him the name Jesus.

Attempts to Recapitulate STAP Cells Fail


Japanese stem cell scientist Haruko Obokata was the first author of two papers that appeared in the journal Nature earlier this year that described the derivation of pluripotent stem cells from mature cells without the use of genetic manipulation. Instead, these cells were subjected to environmental stresses such as physical pressure or exposure to acid that, according to these papers, caused the cells to express genes associated with pluripotency. Culturing of these cells led to the derivation of pluripotent stem cells lines. Thus were born STAP or stimulus-triggered acquisition of pluripotency cells. Needless to say, these results were hailed as a remarkable advance in stem cell biology.

Unfortunately, as soon as the papers were published, several high-level laboratories tried to recapitulate these results and universally failed. Even more troubling were some of the inconsistencies that came to the forefront in the published papers that the reviews had apparently missed or were ignored by the journal. The RIKEN center where this work was done even launched an internal investigation that concluded that Dr. Obokata was guilty of scientific misconduct.  Obokata gave approval to formally retract her Nature papers.  However, the RIKEN Center gave Obokata and her colleagues until the end of November to prove that she could reproduce STAP cell derivation.

Now the jury is in – Obokata has been unable to replicate her results. In the original experiments, Obokata used a gene fusion that caused the cells to glow green if they expressed genes related to pluripotency. In her replication of her original experiments, Obokata produced such green glowing cells when she subjected to environmental stresses. However, this is only a preliminary test that only involved a few such cells. More rigorous tests that were conducted, however, failed. In this case, Obokata’s stressed adult cells were introduced into a mouse embryo to see whether they could contribute to the development of various tissues during animal development. Obokata’s stressed cells, however, were unable to integrate into the developing embryos. Since this is the ultimate test for pluripotency, and since these cells were not able to pass this test, it seems virtually certain that Obokata’s original results were completely bogus.

With her signature conclusions in tatters, Obokata has resigned from the RIKEN center. In a very emotional resignation letter, Obokata wrote she could not “find words enough to apologize… for troubling so many people at RIKEN and other places.”  The RIKEN president, Ryoji Noyori, wrote in an accompanying statement that Dr. Obokata had been subjected to horrible psychological stress as a result of this affair.  Noyori added that he accepted her resignation to hopefully save her from suffering further from a severe “mental burden.”  One the co-authors of the STAP papers, Japanese stem cell scientist Yoshiki Sasai, committed suicide a few weeks after the retraction of the paper.

Hopefully, RIKEN and the other scientists who were involved in this venture move on and continue with the business of pushing back the frontiers of science.  It is entirely possible that intentional fraud was involved, but ultimately, we will never know.  For now, it is clear that sloppiness and a lack of skepticism about one’s own results contributed to this fiasco.  I think most people simply want to put this whole sordid event behind them.  However, there are pointed lessons to be learned and we will be better investigators if we learn them.

For one, peer review is not omnipotent.  Post-publication review is important and will continue to be important.  Secondly, journals need to be willing to solicit outside opinions to ensure the quality of high-level publications.  Third, the majority of scientists publish in journals that most people will never read.  Their work is not glamorous, but instead document tedious, high-quality, detailed, scientific research.  The majority of such work will never appear in Nature or Science or Cell, but that’s alright because good solid research is still good solid research regardless of where it appears.  It is really too bad that the push for high-visibility publications can cause people to publish too quickly before results have been properly vetted.  The STAP episode might be a reminder for journals to take greater care with the review of original research.

Stem Cell Transplants for SCID Children – Earlier is Better


Severe Combined Immune Deficiency or SCID hamstrings the immune system of newborn children and prevents their immune systems from fighting off any diseases. Children born with SCID must be isolated in a germ-free environment and are sometimes called “bubble children,” since they must go out in the open in space suits that purify their air. However, if children with this “bubble boy” disease have a bone marrow transplant, they can survive. When, however is the best time to give these children such a transplant?

A new study by a research group at the Harvard-affiliated Dana Farber/Boston Children’s Cancer and Blood Disorders Center has reviewed the last ten years of data on treating these young patients. According to the conclusions of this study, children with SCID have the best chance of survival if they undergo a bone marrow stem cell transplant as soon after birth as possible. Consequently, genetic screening of newborn babies for SCID should be expanded, since this disorder leaves affected infants so vulnerable to infection that most die within the first year of life if untreated.

This new research was published in the New England Journal of Medicine, and analyzed data on 240 SCID children who underwent transplants at 25 centers across North America between Jan. 1, 2000, and Dec. 31, 2009 (before the U.S. Department of Health and Human Services recommended newborn screening for SCID in 2010). Currently, 21 states and the District of Columbia, which are home to about two-thirds of all babies born in the United States, screen newborns for SCID. Another nine states are expected to implement newborn screening by the end of 2014.

“The best way to identify patients that early when there is no family history of SCID is through newborn screening,” said Sung-Yun Pai, first author on the study. “Survival is much, much better if infants undergo transplant before they turn 3½ months old and before they contract any SCID-related infections,” said Pai. “The best way to identify patients that early when there is no family history of SCID is through newborn screening.”

While patient age was one of the strongest factors determining the survival of SCID infants, this study also showed that the infection status at the time of transplant and the donor source had the strongest impact on transplant outcomes (i.e., five-year survival and successful immune system reconstitution).

Interestingly, the data used in this study also showed that even though SCID is relatively rare, SCID is twice as common as once thought. “Some children who succumbed to unexplained infections probably suffered from SCID,” Pai noted. SCID is estimated to occur in one of every 50,000 births, up from earlier estimates of one in 100,000.

“Time is not the ally of children with SCID,” said Luigi Notarangelo of Boston Children’s Hospital, who was one of the study’s senior authors. Notarangelo is among those who lobbied successfully to establish SCID newborn screening in Massachusetts in 2009. “Because they do not have a functional immune system, the longer the wait before a transplant, the greater the risk they will contract a potentially devastating infection.”

Children who underwent transplant before 3½ months of age had excellent survival, and this was regardless of donor source or infection status. Likewise, children who underwent transplant with stem cells from a tissue-matched sibling donor. Children outside that age group also had very good survival regardless of donor source, but only if the patient did not have an active infection at the time of transplant. The effect of the donor type and pre-transplant conditioning on survival rates was important only in actively infected patients.

Other findings from this study included:
• 74 percent of the 240 patients studied survived at least five years.
• Among patients who underwent transplant at younger than 3½ months, 94 percent survived.
• Virtually all (97 percent) of the patients who received stem cells from a matched sibling donor survived.
• At 50 percent, survival was lowest among patients who were older than 3½ months and had active infections at the time of transplant. Actively infected infants who did not have a matched sibling donor and who received immunosuppressive or chemotherapy before transplant had particularly poor survival rates (39-53 percent).
• Among patients who never had an infection, 90 percent survived, as did 82 percent of patients whose infection had resolved before transplant.

While survivors who received chemotherapy conditioning had stronger immune systems after transplant, it is still unknown if the drugs used to wipe out the patient’s immune systems before the bone marrow transplant pose a long-term risk when given to such young patients.

“This study accomplishes several things,” she said. “First, it creates a baseline with which to compare patient outcomes since the advent of newborn screening for SCID. Second, it provides guidance for clinicians regarding the use of chemotherapy conditioning before transplantation. Third, it highlights the relative impacts of infection status and patient age on transplant success.

“Lastly, it establishes the importance of early detection and transplantation, which points to the benefit of expanding newborn screening for SCID as broadly as possible.”

Americord Registry Funds Research in the Use of Stem Cells for Cancer Patients


Headquartered in New York City, the Americord Registry is one of the leaders in umbilical cord blood, cord tissue and placenta tissue banking. Americord collects, processes, and stores newborn stem cells from umbilical cord blood for future medical or therapeutic use. These uses include the treatment of many blood diseases, including sickle-cell anemia and leukemia.

The Americord Registry has announced that it will fund a research project by the Masonic Cancer Center at the University of Minnesota. This research will examine the potential use of donor stem cells in patients who have been previously treated for three different cancers of the blood or bone marrow; lymphoma, myeloma, or chronic lymphocytic leukemia.

Masonic Cancer Center researchers would like to use donor stem cells to further treat patients who have previously received chemotherapy. Two chemotherapeutic agents, cyclophosphamide and busulfan, for example, arrests the growth of cancer cells, and additionally, prevents the patient’s immune system from rejecting implanted stem cells from a donor. Donated stem cells, for bone marrow or umbilical cord blood, will not share the same array of cell surface proteins as the patient, and might be rejected by the patient’s immune system. However, cancer patients who have been treated with chemotherapeutic agents might be able to tolerate implanted cells, since the anti-cancer drugs might also dull the immune system to the implanted stem cells. These donated stem cells may replace the patient’s immune cells and help destroy any remaining cancer cells.

Americord has a Corporate Giving Program that was established to support research into the therapeutic uses of stem cells from umbilical cord blood, cord tissue, and placenta tissue. The funding for this research comes from Americord’s Corporate Giving Program.

“Americord is committed to supporting the advancement of stem cell treatments and technologies,” said Americord CEO Martin Smithmyer. “We are excited about the research being done at the Masonic Cancer Center and the potential it has for future treatment options.”

The study at the Masonic Cancer Center began in February 2008 and is scheduled to be completed by January 2015. It is registered with ClinicalTrials.gov in accordance with best practices and requirements of the U.S. Food and Drug Administration.

Muscle Wasting in Muscular Dystrophy Due to Defective Muscle Stem Cells, But Can Be Treated with Blood Pressure Drug


By utilizing a mouse model of Duchenne muscular dystrophy (DMD), researchers at Stanford University School of Medicine have compared gene expression differences between muscle stem cells from DMD mice and muscle stem cells from non-DMD mice. Muscle stem cells from DMD mice express connective-tissue genes associated with fibrosis and muscle weakness as opposed to those from non-DMD mice.

DMD mice, just like their human counterparts, experience progressive muscle degeneration and accumulate connective tissue within the muscle as they age. This new study strongly suggests that the stem cells that surround the muscle fibers might be responsible for this defect. During the course of the disease, muscle stem cells in DMD mice become less able to make new muscle and instead begin to express genes involved in the formation of connective tissue. Excess connective tissue causes scarring (a condition called fibrosis), and these excess scars can accumulate in other organs besides muscle, including the lungs, liver and heart. In the skeletal muscles of people with muscular dystrophy, scarring impairs muscle function and leads to increasing weakness and stiffness, which are hallmarks of the disease.

In addition to this discovery, Thomas Rando, professor of neurology at Stanford University Medical School, and his colleagues showed that these abnormal changes in muscle stem cells could be prevented in laboratory mice by giving the animals a drug that is already approved for use in humans. This drug blocks a signaling pathway involved in the development of fibrosis. Of course more work is required, but scientists are hopeful that a similar approach may one day help treat children with muscular dystrophy.

“These cells are losing their ability to produce muscle, and are beginning to look more like fibroblasts, which secrete connective tissue,” said Dr. Rando. “It’s possible that if we could prevent this transition in the muscle stem cells, we could slow or ameliorate the fibrosis seen in muscular dystrophy in humans.”

Rando and his coworkers published their findings in Science Translational Medicine. Rando, who is the senior author of this paper, is also the director of the Glenn Laboratories for the Biology of Aging and is also the founding director of the Muscular Dystrophy Association Clinic at Stanford. Rando’s former postdoctoral scholar Stefano Biressi, who is presently at the Centre for Integrative Biology at the University of Trento in Italy, is the lead author of this paper.

DMD is a truly devastating disease that affects about 1 in every 3,600 boys born in the United States. The hallmark of this disease is the severe, progressive muscle weakness that confines patients to a wheelchair by early adolescence and eventually leads to paralysis. Mutations in the dystrophin gene cause DMD. The dystrophin gene encodes the Dystrophin protein, which connects muscle fibers to the surrounding external matrix, which stabilizes the fibers, enhances their strength and prevents their injury. Mutations in the dystrophin gene cause production of defective copies of the dystrophin protein. Without functional copies of Dystrophin, the unanchored muscle is unstable, weak, and subject to constant injury. DMD patients are almost always boys because the dystrophin gene is located on the X chromosome. Girls must inherit two faulty copies of the dystrophin gene to contract DMD, which is unlikely because male carriers often die in early adulthood.

By decelerating the fibrotic activity of muscle stem cells in DMD patients, it is possible to delay or even fix the scarring observed in human DMD patients. Normally, muscle stem cells are stimulated when muscles are damaged, and they divide into new cells, some of which form new muscle. In DMD mice, however, muscle stem cells the lack a functional copy of the dystrophin gene slowly begin to resemble fibroblasts instead of muscle-making stem cells.

In this study, Biressi and Rando used a strain of laboratory mice in which the muscle stem cells express a glowing protein when they are treated with a drug called tamoxifen. These glowing mice were then mated with another mouse strain that had a defective copy of the dystrophin gene. These DMD mice now had muscle stem cells that glowed when treated with tamoxifen, which allowed Biressi, Rando and others to trace the movements and activities of muscle stem cells. They discovered that the expression of myogenic genes associated with the regeneration of muscle in response to injury was nearly completely lacking in many of the muscle stem cells in the mice after just 11 months. However, the expression of fibrotic genes had increased compared with that of control animals. The muscle stem cells from the DMD animals were also oddly located, since instead of being nestled next to the muscle fibers where they normally are found, they had begun to move away into the spaces between tissues.

Such increased fibrosis is also observed during normal aging and this process is governed by signaling proteins, which include the Wnt and TGF-beta protein families. Wnt plays a critical role in embryonic development and cancer; TGF-beta controls cell division and specialization. Rando and Biressi hypothesized that inhibiting the Wnt/TGF-beta pathway in DMD would inhibit fibrosis in the animals’ muscles.

To do this, they turned to a blood pressure medicine called losartan. Losartan inhibits the expression of the genes for TGF-beta types 1 and 2, and therefore, might interrupt the signaling pathway that leads the muscle stem cells astray. When DMD mice were treated with losartan, the drug prevented the muscle stem cells from expressing fibrosis-associated genes and partially maintained their ability to form new muscle.

“This scar tissue, or fibrosis, leaves the muscle less elastic and impairs muscle function,” Rando said. “So we’d like to understand why it happens, and how to prevent it. It’s also important to limit fibrosis to increase the likelihood of success with other possible therapies, such as cell therapy or gene therapy.”

TGF-beta-1 is an important signaling molecule throughout the body. Therefore, researchers are now working to find ways to specifically inhibit TGF-beta-2, which is involved in the transition of the muscle stem cells from muscle makers to scar producers. They’re also interested in learning how to translate the research to other diseases.

“Fibrosis seems to occur in a vicious cycle,” Rando said. “As the muscle stem cells become less able to regenerate new muscle, the tissue is less able to repair itself after damage. This leads to fibrosis, which then further impairs muscle formation. Understanding the biological basis of fibrosis could have a profound effect on many other diseases.”

Genetically Engineered Pig Provides a Novel Tool to Study Life-Threatening Arrhythmias


A NYU Langone Medical Center research team has developed the first large animal model of an inherited arrhythmic syndrome. This animal model system should help heart scientists parse those mechanisms that establish the heart rhythm and conduct electrical impulses around the heart. This novel pig model can also help in the development of better treatments for inherited forms of life-threatening arrhythmias, which are a significant cause of sudden cardiac death.

These findings were published online in the Journal of Clinical Investigation. Already, this model system helped define what causes lethal arrhythmias in those patients with abnormal cardiac sodium channels. Normal conduction of electrical currents throughout the heart requires the normal functioning of these sodium channels. Disease-causing mutations in cardiac ion channels, which are technically referred to as “channelopathies,” can cause atrial and ventricular arrhythmias, progressive cardiac conduction disorders, and sudden cardiac death.

“By developing a genetically engineered pig sodium channelopathy model, we are now able to examine the mechanisms responsible for lethal arrhythmias in a human-like heart and investigate new therapies aimed at reducing sudden cardiac death,” said lead author David S. Park, MD, PhD, assistant professor, Leon H. Charney Division of Cardiology, Department of Medicine at NYU Langone.

Up to the present time, heart researchers have primarily used cultured heart cells and mouse models to study cardiac arrhythmias in humans. However, rodent hearts are no terrible good model systems for human heart diseases. However, “because of similarities of the pig heart to human hearts, research with the pig model will prove invaluable in gaining further insights into the mechanisms that underlie life-threatening arrhythmias,” said Glenn I. Fishman, MD, the study’s senior author, and Director of the Leon H. Charney Division of Cardiology at NYU Langone.

Both Drs. Fishman and Park envision a future where novel therapies, such as drugs that can enhance cardiac sodium channel expression or radiofrequency ablation procedures, can first be tested in the pig model before application to patients. “A better understanding of arrhythmia mechanisms should yield better therapies in the future,” said Dr. Park.

Three-Dimensional Vaccines Sensitize the Immune System to Cancer


Cancer has the capacity to fool the immune system and evade attack from immune cells. This act of immune system evasion allows tumors to grow and spread throughout the body without any resistance. A relatively new strategy called immunotherapy attempts to stimulate the patient’s immune system to mount an immune response against the tumor and sensitize it to the tumor. However, a new protocol developed by a research teams at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard’s School of Engineering and Applied Sciences (SEAS) uses a three-dimensional structure to program the immune system to attack and destroy tumors.

The senior author of this study, David Mooney, who is a Wyss Institute Core Faculty member and the Robert P. Pinkas Professor of Bioengineering at Harvard SEAS, described this new technique: “We can create 3D structures using minimally–invasive delivery to enrich and activate a host’s immune cells to target and attack harmful cells in vivo.”

This 3-D structure consists of tiny, biodegradable rod–like structures made from silica, known as mesoporous silica rods (MSRs). These MSRs can be loaded with biological and chemical drug components, and then injected by needle just underneath the skin. These rods spontaneously assemble at the injection site to form a three–dimensional scaffold (think of pouring a box of match sticks into a pile on a table). The porous spaces between the MSRs are large enough to recruit and fill with dendritic cells. Dendritic cells are immune cells that play the part of surveillance cells that identify foreign cells and substances and trigger an immune response to those things identified as foreign.

Mesoporous silica rods (MSRs) spontaneously assemble to form a porous 3D scaffold, as seen in this SEM image. The 3D scaffold has many nooks and crannies and is large enough to house tens of millions of recruited immune cells. Credit: Wyss Institute at Harvard University
Mesoporous silica rods (MSRs) spontaneously assemble to form a porous 3D scaffold, as seen in this SEM image. The 3D scaffold has many nooks and crannies and is large enough to house tens of millions of recruited immune cells.
Credit: Wyss Institute at Harvard University

“Nano–sized mesoporous silica particles have already been established as useful for manipulating individual cells from the inside, but this is the first time that larger particles, in the micron–sized range, are used to create a 3D in vivo scaffold that can recruit and attract tens of millions of immune cells,” said co-lead author Jaeyun Kim, Ph.D., an Assistant Professor of Chemical Engineering at Sungkyunkwan University and a former Wyss Institute Postdoctoral Fellow.

MSRs are made in the lab with nanopores, which are small holes that can be filled with specific cytokines, oligonucleotides, large protein antigens, or any variety of drugs. Thus, these structures are excellent repositories that can present a vast range of possible combinations to treat a range of infections or stimulate the immune system to attack several different invading elements.

“Although right now we are focusing on developing a cancer vaccine, in the future we could be able to manipulate which type of dendritic cells or other types of immune cells are recruited to the 3D scaffold by using different kinds of cytokines released from the MSRs,” said co-lead author Aileen Li, a graduate student pursuing her Ph.D. in bioengineering at Harvard SEAS. “By tuning the surface properties and pore size of the MSRs, and therefore controlling the introduction and release of various proteins and drugs, we can manipulate the immune system to treat multiple diseases.”

Once the 3D scaffold has recruited dendritic cells from the body, the drugs contained in the MSRs are released. These drugs activate the dendritic cells and initiate an immune response. The activated dendritic cells then leave the MSR-based scaffolds and travel to the lymph nodes, where they raise alarm and direct the body’s immune system to attack specific cells, such as cancerous cells. Within a few months, the body naturally degrades the MSRs, and they dissolve and leave no trace of their presence.

To date, this team has only tested their 3D vaccines in mice, but these 3D vaccines have proven to be remarkably effective. Injectable 3D scaffolds recruited and attracted millions of dendritic cells in a host mouse, before dispersing the cells to the lymph nodes and triggering a powerful immune response.

These vaccines are easily and rapidly manufactured so that they could potentially be widely available very quickly in the face of an emerging infectious disease. “We anticipate 3D vaccines could be broadly useful for many settings, and their injectable nature would also make them easy to administer both inside and outside a clinic,” said Mooney.

Since the vaccine works by triggering an immune response, the method could even be used preventively by building the body’s immune resistance prior to infection.

“Injectable immunotherapies that use programmable biomaterials as a powerful vehicle to deliver targeted treatment and preventative care could help fight a whole range of deadly infections, including common worldwide killers like HIV and Ebola, as well as cancer,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D. who is also Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at Harvard SEAS. “These injectable 3D vaccines offer a minimally invasive and scalable way to deliver therapies that work by mimicking the body’s own powerful immune–response in diseases that have previously been able to skirt immune detection.”

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.

Repairing Bladders with Bone Marrow Stem Cells and Bladder Acellular Matrix


The bladder is subject to several different types of conditions that can compromise its function. Cancer of the bladder can necessitate its removal. A congenital condition called exstrophy causes the bladder to protrude through a hole in the abdominal wall also requires surgical repair of the bladder. Finally, trauma to the bladder as in the case of trampoline athletes or people who have had surgical damage to the bladder, may also repair bladder repair.

In order to repair the bladder, extra tissue must be added to it. Finding tissue to act as bladder has not been easy. In the past, surgeons have used grafts from skin, bladder submucosa, omentum, dura, peritoneum, colon, small intestine, and synthetic polymers have been used to augment the bladder. All of these compounds have their pluses and minuses for reconstructing a bladder, but all of them do not appropriately recapitulate the mechanical, structural, and functional properties of the bladder.

Currently a surgical procedure called enterocystoplasty is the most effective surgical solution for augmentation of the bladder. This procedure uses a small piece of the large intestine to increase the size of the bladder, and while it certainly improves continence, it has several complications associated, which include, metabolic disturbances, urinary stones (urolithiasis), increased mucus production, infections and increased risk of cancer of the bladder. Is there a better way?

A collaborative research project between Daniel L. Coutu from ETH Zürich, in Basel, Switzerland, Wally Mahfouz, Oleg Loutochin, and Jacques Corcos from McGill University, in Montreal, Canada, Wally Mahfouz from Alexandria University in Alexandria, Egypt, and Jacques Galipeau from Emory University in Atlanta, Georgia examined the use of bladder a cellular matrices or BAMs in combination with bone marrow-derived mesenchymal stromal cells to repair bladders in laboratory animals.

BAMs consist of bladders from animals that have been completely stripped of their cells with detergents and enzymes. Once all the cells and cell remnants have been removed, these BAMs can be molded into the form of a bladder, after which cells are reapplied. BAMs contain all the chemical nooks and crannies for cells to find, attach to, then and differentiate. Also, the bladder is a relatively simple tissue in that it has an inner epithelium (urothelium) that sits on a basement membrane, a smooth muscle layer surrounding the urothelium, and an outer serosa layer that is an extension of the peritoneum that it covered by an adventitia of connective tissue. A bladder matrix devoid of cells has all the right structures for cells to occupy, bit it needs to be repopulated with cells.

Corcos and his collaborators purchased pig bladders from slaughter houses and subjected them to detergents and enzymes until no cells were left on them. Then they used mesenchymal stromal cells from the bone marrow of rats to reseed the bladders with new cells. These structures were then used to repair the bladders to laboratory rats whose bladders had been partially removed. All the animals were then tested for retention of urine, muscle tone, pressure tolerance, and other indicators of bladder function.

The results of these tests demonstrated that the engineered bladders not only worked, but worked quite well. Some animals only received the BAM without cells, and these animals had bladders that worked better than nothing, but not terribly well. However, the animals that received BAM + mesenchymal stem cells had bladders that, for all intents and purposes, showed normal function by 6 months after the procedure.

Another significant finding of this study is that none of the animals that received BAMs had to be given any anti-rejection drugs. The immune systems of these animals did not reject the animal-based matrices.

Finally, post-mortem examinations of these animal bladders established that smooth muscle regeneration and nerve and blood vessels regeneration was also robust in these animals.

Before this procedure can work in humans, it will need to work in larger animal systems. Rat urinary systems are similar to humans, but not the same. The large advance in this study is the observation that internal bladder tissues such as smooth muscle, nerves and blood vessels can be regenerated with mesenchymal stem cells. These stem cells probably secrete a variety of molecules that promote the growth of blood vessels and nerves into the bladder.

In the words of these authors: “we demonstrated the in vivo superiority of MSCs-seeded BAMs compared with unseeded BAMs in bladder tissue engineering. Our approach is fully translatable to large animals and humans, where autologous MSCs could be seeded on allogeneic, cadaveric or xenogenic BAMs. The method presented here is a viable alternative to current treatment modalities and should prevent most complications associated with them. This study demonstrates the superiority of MSCs-seeded BAM compared to BAM alone in bladder augmentation and provides a strong basis to test our novel approach in large animal models and eventually in humans.”

Amen.

3-D Printed Meniscus Regenerated Meniscus in Sheep


Within the knee-joint, on either side, is a cartilage shock absorber called the meniscus. Tears to this structure can cause pain and swelling in the knee and erosion of the meniscus can lead to bone-on-bone joints that abrade the bone and cause further inflammation and osteoarthritis. Because the meniscus is made of cartilage, and since cartilage can be grown in the laboratory, it should be possible, in theory, to make a new meniscus. Researchers at Columbia University Medical Center have succeeded in using 3-D printing to do that just.

The laboratory of Jeremy Mao used made personalized 3-D implants made from a scaffold infused with human growth factors. When implanted into the knee, these growth factors stimulate the body to regenerate the meniscus on its own. Mao and his coworkers successfully tested their treatment strategy in sheep. Their procedure could provide the first effective and long-lasting way to repair of damaged menisci, which occur in millions of Americans each year and can lead to debilitating arthritis. This work from the Mao lab was published in Science Translational Medicine.

“At present, there’s little that orthopedists can do to regenerate a torn knee meniscus,” said Mao, who is the Edwin S. Robinson Professor of Dentistry (in Orthopedic Surgery) at the Medical Center. “Some small tears can be sewn back in place, but larger tears have to be surgically removed. While removal helps reduce pain and swelling, it leaves the knee without the natural shock absorber between the femur and tibia, which greatly increases the risk of arthritis.”

Heavily damaged menisci can be replaced with a meniscal transplant that utilizes tissue from other parts of the body or from cadavers. Such transplants, however, have a low success rate and carries significant risks. Approximately one million meniscus surgeries are performed in the United States each year.

Mao and his colleagues began with MRI scans of the intact meniscus in the undamaged knee. Special computer software then converts these high-resolution scans in to a 3D image. Data from these images are then used to drive a 3D printer, which produces a scaffold in the exact shape of the meniscus, all the way down to a resolution of 10 microns, which is less than the width of a human hair. The scaffold takes about 30 minutes to print and is made from an organic polymer called polycaprolactone, which is the same biodegradable polymer used to make surgical sutures.

The printed scaffold is infused with two recombinant human proteins: connective growth factor (CTGF) and transforming growth factor β3 (TGFβ3). In earlier work, Mao’s team discovered that sequential delivery of these two proteins attracts resident stem cells from the body and induces them to form meniscal tissue.

In order for a meniscus to properly form, these growth factors must be released from specific areas of the scaffold and in a specific order. To accomplish this, the growth factors were encapsulated in two types of slow-dissolving polymeric microspheres. The first of these microspheres released CTGF, which stimulates the production of the outer meniscus. The second microspheres release TGFβ3, which induces the production of the inner meniscus. Finally, this protein-infused scaffold is inserted into the knee so that it can direct the generation of a new meniscus. When these printed, growth factor-infused scaffolds were implanted into the knees of sheep, the meniscus regenerated in approximately four to six weeks. The implanted, biodegradable scaffold eventually disintegrates.

“This is a departure from classic tissue engineering, in which stems cells are harvested from the body, manipulated in the laboratory, and then returned to the patient—an approach that has met with limited success,” said Mao. “In contrast, we’re jumpstarting the process within the body, using factors that promote endogenous stem cells for tissue regeneration.”

“This research, although preliminary, demonstrates the potential for an innovative approach to meniscus regeneration,” said co-author Scott Rodeo, sports medicine orthopedic surgeon and researcher at Hospital for Special Surgery in New York City. “This would potentially be applicable to the many patients who undergo meniscus removal each year.”

Mao and others tested their procedure in 11 sheep. Even though they are four-legged creatures, sheep knees closely resemble that of humans, and therefore, as an excellent model system for orthopedic research. These animals were randomized to have part of their knee meniscus replaced with a protein-infused 3D scaffold (the treatment group) or a 3D scaffold that was not infused with growth factors (the nontreatment group). After three months, the treated animals all walked normally. A postmortem analysis of the treated animals demonstrated that the regenerated meniscus in the treatment group had structural and mechanical properties very similar to those of natural meniscus. Mao’s laboratory is now conducting studies to determine whether the regenerated tissue is long-lasting.

“We envision that personalized meniscus scaffolds, from initial MRI to 3D printing, could be completed within days,” said Mao. The personalized scaffolds will then be shipped to clinics and hospitals within a week. The researchers hope to begin clinical trials once funding is in place.

“These studies provide clinically valuable information on the use of meniscal regeneration in the knees of patients with torn or degenerate menisci,” said co-author Lisa Ann Fortier, professor of large animal surgery at Cornell University College of Veterinary Medicine in Ithaca, N.Y. “As a veterinary orthopedic surgeon-scientist on this multi-disciplinary team, I foresee the added bonus of having new techniques for treating veterinary patients with torn knee meniscus.”

Heart Muscle Cells Produced from Induced Pluripotent Stem Cells Repair Heart Attacks in Pigs


When heart muscle cells are made from embryonic stem cells, they integrate into the heart and form proper connections with other heart muscle cells. Such experiments have been conducted in mice, guinea pigs, and nonhuman primates (i.e. monkeys). Chong and others earlier this year (Nature (2014) 510, 273-277) implanted heart muscle cells produced from embryonic stem cells into the hearts of nonhuman primates that had suffered from heart attacks. There was extensive evidence of engraftment of these cells, remuscularization of the heart, and electrical synchronization 2 to 7 weeks after transplantation. However, despite these successes, the hearts of some of these animals also showed abnormal heart beat patterns (known as arrhythmias). Such a problem has also been observed in other laboratory animals as well (see my book The Stem Cell Epistles), and this problem has to be addressed before derivatives of pluripotent stem cells can be used to treat damaged hearts (pluripotent means capable of differentiating into all the mature adult cell types).

Jianyi Zhang and his colleagues at the University of Minnesota have used induced pluripotent stem cells made from human skin cells to produce heart muscle cells that were used to treat pigs that had suffered from induced heart attacks.  Their results differed slightly from those of Chong and others.

Zhang and others noted that implanted heart muscle cells typically survive better if they are implanted with blood vessel cells (endothelial cells or ECs).  This was first shown in culture by Xiong and others in 2012 (Circulation Research 111, 455-468), but other work has confirmed this.  That is, Zhang’s coworkers in his laboratory co-transplanted heart muscle cells made from induced pluripotent stem cells with endothelial cells and smooth muscle cells (which are also a part of blood vessels), and saw that the co-transplanted cells survived much better than heart muscle cells that were transplanted without these other cell types.

On the basis of these experiments, Zhang and his crew decided that implanted heart muscle cells would do much better if they were implanted into pig hearts if they were implanted with endothelial and smooth muscle cells.  This was the hypothesis that Zhang and others wanted to test in this paper (which was published in Cell Stem Cell, Dec 4, 2014, 750-761).

Skin biopsies from human volunteers were used as a source of skin cells that were then genetically engineered and then cultured to form human induced pluripotent stem cells (hiPSCs).  These cultured hiPSCs were differentiated into heart muscle cells by means of the “Sandwich method,” which yielded beating heart muscle cells in about 30 days.  Additionally, their hiPSC lines were differentiated into smooth muscle and endothelial cells as well.

Next, Zhang and his colleagues and collaborators used 92 pigs and subjected them to experimentally-induced heart attacks.  Why pigs?  Pigs are a larger animal than rodents, and their hearts are larger and beat much slower than the hearts of rats and mice.  Therefore, they are a more expensive, but better experimental model system for the human heart.  Nevertheless, these pigs were divided into six different groups (3 pigs died from the procedure, so there were 89 pigs involved in this experiment).  Animals in the first group or SHAM group underwent the surgery to induce a heart attack, but no heart attack was induced.  The second group was called the MI group and this group received no other interventions after surgery.  The Patch group received a fibrin patch over the site of injury, but no cells.  The CM + EC + SMC group received injections of 2 million heart muscle cells, two million endothelial cells, and two million smooth muscle cells directly into the injured portion of the heart.  The Cell + Patch group received all three cell types in a fibrin patched that was imbued with a growth factor called Insulin-like growth Factor-1 (IGF-1) that had been loaded into microspheres.  This causes the growth factor to be released gradually and exert its effects over a much greater period of time.

That’s a lot of information so let’s review – six groups: 1) SHAM (no heart attack; 2) MI (heart attack and no treatment); 3) Patch (just the fibrin patch); 4) Cells + Patch (fibrin patch with the three cell types); 5) Cells (cells, but no patch), and a final group cells Patch + CM (just heart muscle cells in the patch).

Animals were evaluated one week after the heart attack and four weeks after their heart attacks. I am uncertain how soon after the heart attack the treatments were given, but in the paper it reads to me as though the treatments were given right after the heart attacks had been induced.  Because all implanted cells were engineered to glow in the dark, the number of surviving cells could be counted and tracked.

Only 4.2% of the cell survived in the Cells group, up to 9% of the cells in the Cell + Patch group survived.  32% of the cells in the CM + Patch group survived.  Thus, it seemed as though the presence of the other cell types did increase the survival of the heart muscle cells and the patch also increased cell survival rates.  Secondly, the heart function of all the treated groups was better than the MI group, but the hearts treated with Cells + Patch were clearly superior to all the others, with the exception of the SHAM group.  The hiPSC-derived heart muscle cells also clearly engrafted into the hearts of the pigs, but the big surprise in this paper is that THERE WERE NO INDICATIONS OF ARRHYTHMIAS!!!  Apparently the manner in which these hiPSC-derived heart muscle cells integrated and adapted to the native heart in such as way as to preclude irregular electrical activity.  Another indicator measured was ratio of phosphocreatine to ATP.  If that sounds like a language from outer space, it simply means a measurement of the efficiency of muscle mitochondria (the part of the cell that makes all the energy).  Again the Cells + Patch hearts had significantly more efficient mitochondria, and, hence, better energy production than the other hearts.  Damage to mitochondria also tends cause cells to up and die, which means that these cells were in better health that those from the MI group.

This paper shows that an ingenious tissue engineering innovation that uses a fibrin patch and a a combination of cells, not just heart muscle cells can significantly increase the healing after a heart attack.  Also, even though neither embryonic stem cell-derived cells nor iPSC-derived cells are ready for clinical trials, this paper shows that iPSCs are not as far behind iPSCs as some authors have suggested.  Furthermore, because iPSCs would not be subject to immunological rejection, they have an inherent superiority over embryonic stem cells.  The problem comes with the time required to make iPSCs and then derived heart muscle cells from them, which might put it outside the time window for treat of an acute heart attack.

Fingernail Stem Cell Population Identified


The ability of fingernails to grow back, unlike other body parts seems to be the result of the presence of a resident stem cells population.

Researchers at the University of Southern California (USC), led by Krzysztof Kobielak has identified a new stem population in nails that can either self-renew or differentiate into other distinct cell types.

Identifying these cells was no small chore, and Kobielak used an ingenious new technique for attaching fluorescent dyes and other tags to mouse nail cells. while many cells in the nail divided and spread throughout the nail, another small population stayed at the base of the nail and divided slowly or not at all.

Localization of LRCs in the nail proximal fold. (A) Components of the mouse nail. Top view, horizontal sections of the fingertip before (C) and after (B and D) 4 wk of chase with Dox identifying a population of H2BGFP marked LRCs surrounding the nail structure. Side view, perpendicular sections of the digit tip before (E and G) and after (F) 4 wk of chase with Dox demonstrating the presence of upper LRCs in the upper PF. (H) Lower LRCs at the lower nail PF. GFP; green fluorescent protein; HF, hair follicle; L-LRCs, lower label-retaining cells; U-LRCs, upper label-retaining cells. Yellow box denotes region of interest in B, and red and blue boxes (G) denote representative U-PF and L-PF regions for orientation. (Scale bars: 50 μm.)
Localization of LRCs in the nail proximal fold. (A) Components of the mouse nail. Top view, horizontal sections of the fingertip before (C) and after (B and D) 4 wk of chase with Dox identifying a population of H2BGFP marked LRCs surrounding the nail structure. Side view, perpendicular sections of the digit tip before (E and G) and after (F) 4 wk of chase with Dox demonstrating the presence of upper LRCs in the upper PF. (H) Lower LRCs at the lower nail PF. GFP; green fluorescent protein; HF, hair follicle; L-LRCs, lower label-retaining cells; U-LRCs, upper label-retaining cells. Yellow box denotes region of interest in B, and red and blue boxes (G) denote representative U-PF and L-PF regions for orientation. (Scale bars: 50 μm.)

Kobielak and his team showed that these slow-dividing cells normally contribute to the growth of her nails and nearby skin. However, if the nail undergoes some kind of injury or physical insult, a signaling protein called bone morphogen protein or BMP signals to the nail bed stem cells to switch to exclusively repairing the nail. Thus this nail bed stem cell population has the flexibility to perform dual roles in the finger tips.

Nail proximal fold cells participate in nail regeneration in response to plucking injury and upon transplantation. (A) Whole-mount Tomato expression in regenerated nails 2 wk after plucking. Linear streams of Tomato+ cells (red) in regenerating nails (arrows) extending from the nail base toward the tip. (B) Schematic model representing the role of K15+ NPFSCs during nail regeneration. (C–C′′) K5 expression (green) in regenerating nails localizing the linear K15-derived, Tomato+ (red) cell streams emanating from the basal K5+ Mx extending upward (arrows) into the overlying differentiated NP; yellow box denotes region of interest in (C′ and C′′). (D) H2BGFP+ nail LRCs persist in the finger following nail removal. (E) Nail LRCs are quiescent, whereas the nail Mx contains actively proliferating cells marked by BrdU incorporation. (F) Upon NP removal, LRCs become activated, indicated by Ki67 coexpression. (G) Nail LRCs transplantation strategy. H2BGFP+ nail cells contribute to the nail structure 17 d after transplantation (H), sectioning of 22-d chased transplant (I), demonstrating the presence of remaining LRCs from the transplant. d, day; Epi, epidermis; GFP, green fluorescent protein; L-LRCs, lower label-retaining cells; U-LRCs, upper label-retaining cells. DAPI counterstaining (blue) was used to localize cell nuclei in immunofluorescent images. (Scale bars: A and H, 500 μm; C–F and I, 50 μm.)
Nail proximal fold cells participate in nail regeneration in response to plucking injury and upon transplantation. (A) Whole-mount Tomato expression in regenerated nails 2 wk after plucking. Linear streams of Tomato+ cells (red) in regenerating nails (arrows) extending from the nail base toward the tip. (B) Schematic model representing the role of K15+ NPFSCs during nail regeneration. (C–C′′) K5 expression (green) in regenerating nails localizing the linear K15-derived, Tomato+ (red) cell streams emanating from the basal K5+ Mx extending upward (arrows) into the overlying differentiated NP; yellow box denotes region of interest in (C′ and C′′). (D) H2BGFP+ nail LRCs persist in the finger following nail removal. (E) Nail LRCs are quiescent, whereas the nail Mx contains actively proliferating cells marked by BrdU incorporation. (F) Upon NP removal, LRCs become activated, indicated by Ki67 coexpression. (G) Nail LRCs transplantation strategy. H2BGFP+ nail cells contribute to the nail structure 17 d after transplantation (H), sectioning of 22-d chased transplant (I), demonstrating the presence of remaining LRCs from the transplant. d, day; Epi, epidermis; GFP, green fluorescent protein; L-LRCs, lower label-retaining cells; U-LRCs, upper label-retaining cells. DAPI counterstaining (blue) was used to localize cell nuclei in immunofluorescent images. (Scale bars: A and H, 500 μm; C–F and I, 50 μm.)

The members of the Kobielak laboratory are also interested in other types of signals and what they might do to these nail bed stem cells. For example, could they induce them to differentiate into additional cell types besides skin and nail? Could they aid in amputation repair and the repair of severe skin injuries?

Kobielak said: “That was very surprising discovery [sic], since the dual characteristics of these nail stem cells to regenerate both the nail and the skin under certain physiological conditions is quite unique and different from other skin stem cells, such as those of the hair follicle or sweat gland.”

Growing Human Esophagus Tissue from Human Cells


Tracy Grikscheit of the Saban Research Institute of Children’s Hospital Los Angeles and her colleagues have successfully grown a tissue engineered esophagus on a relatively simple biodegradable scaffold after seeding it with the appropriate stem and progenitor cells.

Progenitor cells have the ability to differentiate into specific cell types and can migrate to a particular target tissue. Their differentiation potential depends on the parent cell type from which they descended and their “niche” or local surroundings. The scaffold upon which these cells were seeded is composed of a simple polymer, but interestingly, several different combinations of cell types were able to generate a replacement organ that worked well when transplanted into laboratory mice.

“We found that multiple combinations of cell populations allowed subsequent formation of engineered tissue. Different progressive cells can find the right “partner” cell in order to grow into specific esophageal cell types; such as epithelium, muscle or nerve cells, and without the need for exogenous growth factors. This means that successful tissue engineering of the esophagus is simpler than we previously thought,” said Grikscheit.

Videos published the paper show a network of muscle cells properly wired with nerves that properly self-organizes whose muscles spontaneously contract.  Such structures are called an esophageal organoid unit (EOU) in culture. Spontaneous contraction is observed within these EOUs.

This study could be the impetus for clinical procedures that can treat children born with portions of their esophagus missing. Since the esophagus carries liquids and food to the stomach from the mouth, it is a vitally important part of the body.

This protocol, could also be applied to patients who have suffered from esophageal cancer and had to have their esophagus removed. Esophageal cancer is one of the fastest growing types of cancer in the United States to date. Alternatively, people who have accidentally swallowed caustic liquids may also benefit from this type of esophageal repair.

This simple scaffold made of a polyglycolic acid/poly-L-lactic acid and coated with the protein collagen is inexpensive and versatile and completely sufficient for the growth of tissue-engineered esophagi from human cells, according to this study. When established in culture, this system can also serve as a model system to study the cell dynamics and physiology of the esophagus.

A deeper understanding of how esophageal cells behave in response to injury and how various donor cells could potentially expand the pool of potential donor cells for engineered tissue.

Even though this technique has only been tested in animals to date, fine-tuning of this technique might very well ready it for clinical trials in the future.