Artificial Organs that Fit in Your Hand


New technologies are now available that allow scientists to make mock human organs that can fit in the palm of your hand. These organs-on-a-chip can help test drugs and provide excellent model systems for organ function when they are healthy and when they are diseased.

Think of it: a gut-on-a-chip being developed at the Johns Hopkins School of Medicine can help determine if your heart medicine is actually causing your upset stomach or your diet. This type of technology is a high-tech approach to dealing with a scourge of the low-tech world.

“I’m interested in solving a worldwide problem of diarrheal diseases,” says Dr. Mark Donowitz, who runs this lab and studies diarrheal diseases. According to Donowitz, 800,000 children a year die from diseases like cholera, rotavirus and certain strains of E. coli.

“We’ve failed so far to find drugs to treat diarrhea using cell culture models and mouse intestine,” Donowitz says. Unfortunately. mouse digestive systems don’t react the way human s do to these germs. Therefore, they aren’t very helpful for studying diseases of the gut. Therefore, Donowitz’s team is building his gut-on-a-chip technology in what he hopes will be a superior technique for studying these these diseases.

Postdoctoral researcher Jennifer Foulke-Abel holds the gut-on-a-chip inside the lab at Johns Hopkins School of Medicine.
Postdoctoral researcher Jennifer Foulke-Abel holds the gut-on-a-chip inside the lab at Johns Hopkins School of Medicine.

When you hold one of these devices in the palm of your hand, it is little more than a thin sheet of glass, topped with a plastic microscope slide with a tiny cavity inside. Half a dozen spaghetti-size tubes extend from the device.

“The reason there are so many tubes is we have a vacuum chamber that will cause the membrane to stretch, the way the intestine stretches as it moves food along,” Fouke-Abel explains.

Cells isolated from a human intestine are placed into a tiny chamber around that membrane, and the cells divide, grow and organize themselves into a small version of part of a human gut. The device, when operating, might hold 50,000 gut cells.

The first step of this research is to determine if the cells in the chip react the same way to diseases as cells in the human gut.

“And in all three of the diseases I mentioned, we’ve been able to take that first step,” Donowitz says. “So we know that these appear to be really good models of the human disease.”

To date, the guts-on-a-chip produce digestive enzymes, hormones and mucus, but they don’t yet incorporate other parts of the human intestine, such as blood vessels or nerve cells.

“They all have to be incorporated if you want to move from a simple to a more complex system, which I think you need to do if you are going to reproduce intestinal biology,” Donowitz says.

However, Donowitz’s laboratory is moving in that direction. Once it has built a complete system, they will use it to test potential drugs for the diseases being studied. “We think this could be a real step forward in terms of reducing waste-of-time drug development,” Donowitz says.

While the Donowitz lab at Johns Hopkins is working to develop the gut, other labs scattered around the country are working on other organ systems.

“There’s going to be a brain-on-a-chip, liver, heart and so on,” says Danilo Tagle, who coordinates this overall effort at the National Center for Advancing Translational Sciences, which is part of the National Institutes of Health. The grant structure for this study section will fund the development of 10 organ systems in all.

“The goal is actually to tie them in all together,” Tagle says. To this end, the mini-organs on a chip will collectively work together, much like an entire human being on a chip.

Tagle’s hope is that scientists can build many of these systems, each one based on the cells from an individual person. This would create an array of cell-based stand-ins for research or even diagnoses.

“And so you can identify which part of the population might be more responsive to particular drugs, or identify a subset of the population that might be more vulnerable to the harmful effects of a particular drug,” Tagle says.

According to Tagle, this $75 million, five-year project took off thanks to pioneering work at the Wyss Institute for Biologically Inspired Engineering at Harvard. The research has been so promising, Wyss spun off a private company to pursue it.

“It’s called Emulate,” says Donald Ingber, founding director of the Wyss Institute. “It’s just getting its feet on the ground. We have almost 20 people out of the Wyss Institute who are moving out with it.”

Ingber says it would be too much to expect this technology to replace mice in medical research anytime soon. But he is hoping that this will speed up drug development and make it less expensive, “because if we can identify things that are more likely to work in humans, that’s going to have major impact.”

And there are so many avenues to pursue, he says, there’s plenty of room for both industry and academics to work on building and improving these organs-on-a-chip.

Living Cell Technologies Completes Cell Implants into Parkinson’s Patients


Living Cell Technologies (LCT) is a Australasian biotechnology company with offices in Australia and New Zealand. One of the products pioneered by LCT is NTCELL; a capsule coated with alginate (a porous compound extracted from seaweed) that contains clusters of choroid plexus cells from newborn pigs. NTCELL transplantation allows them to function as a biological factory that produces growth factors and other small molecules that promote new central nervous system growth and repair disease induced by nerve degeneration.

The choroid plexus is the structure in the brain that produces cerebrospinal fluid. These cells also filter wastes from the brain and keeps the brain free of debris and other potentially deleterious material. Choroid plexus cells not only produce cerebrospinal fluid, but also a range of neurotrophins (nerve growth factors) that have been shown to protect against neuron (nerve) cell death in animal models of disease.

Several papers have reported on the use of implanted NTCELL capsules in animal model systems. Luo and others used NTCELLs in nonhuman primates that suffered from chemically induced Parkinson’s disease. This paper reported that the transplanted encapsulated choroid plexus clusters significantly improved neurological functions in these monkeys with Parkinson’s disease (J Parkinsons Dis. 2013 Jan 1;3(3):275-91). An earlier paper also showed that implanted improved the neurological function of rodents with a chemically induced form of Huntington’s disease (Borlongan CV and others, Cell Transplant. 2008;16(10):987-92).

On the strength of these successful animal studies, LCT launched human clinical trials in patients with Parkinson’s disease. On December 15th of last year, LCT announced that the final patient had been successfully implanted in its Phase I/IIa clinical trial of regenerative cell therapy NTCELL for Parkinsons disease. These implantations required a minor surgical procedure, which took place at Auckland City Hospital

This Phase I/IIa clinical trial is being led by Dr. Barry Snow, and is an open-label investigation of the safety and clinical effects of NTCELL in Parkinson’s patients who no longer respond to current therapy. Dr. Snow is the leader of the Auckland Movement Disorders Clinic at the Auckland District Health Board but is also an internationally recognized clinician and researcher in Parkinson’s disease.

These patients will be carefully tracked for improvements in the control of movement and balance. LCT hopes to present the results on this clinical trial, which will last 29 weeks) at the 19th International Congress of Parkinson’s Disease and Movement Disorders in San Diego in June 2015.

Dr Ken Taylor, chief executive, notes that the success of the implant procedure means that the time scale for the LCT clinical program remains intact.

“The treatment phase of the trial has been completed on schedule. We believe NTCELL has the potential to be the first disease-modifying treatment for patients who are failing the current conventional treatment for Parkinson’s disease,” said Dr Taylor.

Even though this Phase I/IIa clinical trial is meant to test the efficacy of NTCELL in Parkinson disease patients, NTCELL also has the potential to be used in a number of other central nervous system indications such as Huntington’s, Alzheimer’s and other types of diseases that affect motor neurons.

Derivation and Culture of Induced Pluripotent Stem Cells on an Artificial Substrate


When mouse embryonic stem cells were first derived in 1981 independently by Gail Martin at UCSF and Evans and Kaufman at Cambridge University, the inner cell mass cells from the blastocyst-stage mouse embryos were cultured on a layer of mouse skin cells that had been treated with a drug that prevented them from dividing or with radiation that did the same. These single layers of mouse skin fibroblasts secreted growth factors that prevented the embryonic stem cells from differentiating and drive them to divide. These layers of cells were known as “feeder” cells, because the secretions of the cells fed the growing embryonic stem cells.

When James Thomson at the University of Wisconsin derived the first human embryonic stem cell lines in 1998, he also used mouse feeder cells to keep the cells growing and undifferentiated. Once the embryonic stem cells were taken from this culture system, they began to differentiate.

Human and mouse embryonic stem cells. (A) Colony of Human Embryonic Stem Cells (Cat# GSC-1103) growing on mitotically arrested feeder layers (Cat Nr GSC-6001M); colony morphology is characteristic of undifferentiated human ES cells. (B) Mouse Embryonic Stem Cells (Cat# GSC-5002) in culture.
Human and mouse embryonic stem cells. (A) Colony of Human Embryonic Stem Cells (Cat# GSC-1103) growing on mitotically arrested feeder layers (Cat Nr GSC-6001M); colony morphology is characteristic of undifferentiated human ES cells. (B) Mouse Embryonic Stem Cells (Cat# GSC-5002) in culture.

However, it became equally clear that using mouse feeder cells represented a problem if human embryonic stem cells were going to be used for clinical purposes because animal cells can harbor occult viruses and other infectious agents that can infect human cells. Also, animal cells possess unusual sugars that are transferred to human cells when they are together in culture. Such foreign sugars can elicit robust immune responses against the cells if they are used for clinical purposes See Martin et al., Nature Medicine 2005; 11:228-232; and Stacey et al., Journal of Biotechnology 2006;125:583-588). Therefore, it became clear that finding ways to grow embryonic stem cells in the absence of feeder lines was an important goal if these cells were going to be used for clinical purposes.

Several laboratories successfully derived so-called “Xeno-free” embryonic stem cells by using protein substrata to grow the cells. These protein substrata included matrigel (animal), human laminin, E-cadherin, and vitronectin (see Xu C,, et al (2001) Nat Biotechnol 19:971–974; Miyazaki T,, et al. (2008) Biochem Biophys Res Commun 375:27–32; Nagaoka M,, et al. (2010) BMC Dev Biol 10:60; Chen G,, et al. (2011) Nat Methods 8:424–429). When Yamanaka and his colleagues discovered procedures for making human induced pluripotent stem cells, once again, feeder lines were initially used, but feeder-free protocols were also developed for deriving xeno-free induced pluripotent stem cells (iPSCs; see Chen G,, et al. (2011) Nat Methods 8:424–429; Nakagawa M,, et al. (2014) Sci Rep 4:3594).

A new report from Luis Gerardo Villa-Diaz, Jin Koo Kim, Joerg Lahann, and Paul H. Krebsbach from the University of Michigan, Ann Arbor, Michigan, has described a way to derive and grow human iPSCs on a completely synthetic substratum. This substratum, poly2-(methacryloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide, or PMEDSAH, forms a hydrogel that is completely synthetic. Therefore, the cells do not touch anything made from genetically manipulated cells or animal products.

Krebsbach and his group used fibroblasts from human gum tissue as their cell source. These cells were reprogrammed into iPSCs by means of infection with recombinant Sendai viruses. These viruses cause expression of the four genes required for reprogram cells (Oct4, Klf4, Sox2, and c-Myc), but they do not insert their viral genomes into the chromosomes of the host cell. Therefore, these viruses only express the reprogramming factors transiently, and afterwards, no trace of them can be found in the iPSC line, provided you properly screen for the absence of the virus.  The reprogrammed cells were grown on the PMEDSAH and the cells not only were reprogrammed on this substratum, but also grew on it rather well.

The gum-based fibroblasts were nicely reprogrammed and made iPSCs that expressed all the right genes and produced tumors called teratomas when implanted into nude mice. The teratoma-production assay is an important test for pluripotency, because teratomas are tumors that consist of the mishmash of different tissue types. The fact that implanted cells produce these tumors with a mixed cell population of such wildly different cell types is an important indication of their pluripotency.

Evaluation of pluripotency of human induced pluripotent stem cells (iPSCs) derived and cultured on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The pluripotency of the three human iPSCs derived and cultured on PMEDSAH was tested by embryoid body (EB) formation, directed in vitro cell lineage differentiation, and teratoma induction. (A): Representative micrograph of EBs from human foreskin fibroblast induced pluripotent stem cells 9 months after derivation and continuous in vitro culture. (B): Graph showing relative RNA transcription levels of genes expressed in cells after directed in vitro differentiation of human iPSCs on PMEDSAH-GPs. (C–E): Representative micrographs of directed in vitro cell lineage differentiation on PMEDSAH-GPs of human iPSCs 9 months after derivation and continuous in vitro culture. Neural differentiation (ectoderm) was achieved after treatment with Noggin (B, C). Definitive endoderm/pancreatic differentiation was induced by activin A treatment (B, D). Mesoderm lineage was obtained after treatment with activin A and BMP4 to induce cardiac muscle differentiation (B, E). Teratoma formation was performed 6 months after derivation and continuous in vitro culture of human iPSCs. (F–H): Representative micrographs of neurons (F), gut glandular epithelium (G), and cartilage (H) identified in teratomas. Scale bars = 200 μm (A), 100 μm (C–E), and 50 μm (F–H). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.
Evaluation of pluripotency of human induced pluripotent stem cells (iPSCs) derived and cultured on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The pluripotency of the three human iPSCs derived and cultured on PMEDSAH was tested by embryoid body (EB) formation, directed in vitro cell lineage differentiation, and teratoma induction. (A): Representative micrograph of EBs from human foreskin fibroblast induced pluripotent stem cells 9 months after derivation and continuous in vitro culture. (B): Graph showing relative RNA transcription levels of genes expressed in cells after directed in vitro differentiation of human iPSCs on PMEDSAH-GPs. (C–E): Representative micrographs of directed in vitro cell lineage differentiation on PMEDSAH-GPs of human iPSCs 9 months after derivation and continuous in vitro culture. Neural differentiation (ectoderm) was achieved after treatment with Noggin (B, C). Definitive endoderm/pancreatic differentiation was induced by activin A treatment (B, D). Mesoderm lineage was obtained after treatment with activin A and BMP4 to induce cardiac muscle differentiation (B, E). Teratoma formation was performed 6 months after derivation and continuous in vitro culture of human iPSCs. (F–H): Representative micrographs of neurons (F), gut glandular epithelium (G), and cartilage (H) identified in teratomas. Scale bars = 200 μm (A), 100 μm (C–E), and 50 μm (F–H). Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Even more importantly perhaps is the genetic integrity of these cells. Karyotypes of these iPSC lines (karyotypes lay our the chromosomes of the cell to see if there are a normal number of chromosomes and if the chromosomes appear normal) revealed that they were beautifully normal. However, hCGH (array-based comparative genomic hybridization) analysis, which uses specific chromosome-specific probes to finding missing or duplicated bits of chromosomes that are too small to see in a karyotype revealed a few missing and added bits to the genomes of all three derived iPSC lines. None of these were in known cancer loci.  As shown in the figure below, these lines had only a few mutations.  The karyotypes used cells from nine months after their derivation.  Thus these cells proved to be rather stable.

Genetic stability of the human iPSCs derived on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The genomic stability of the three human iPSC lines derived on PMEDSAH-GPs was tested 9 months after derivation and continuous in vitro culture. (A): Representative standard G-banding metaphase karyotyping of one of the three human iPSCs derived on PMEDSAH-GPs showing normal male karyotype. (B): Ideogram summarizing chromosome losses and gains (left and right, respectively) of the three human iPSCs as detected by high-resolution array-based comparative genomic hybridization. No mutations are localized in chromosome loci where genes related to stem cells, cancer, or culture adaptation are localized. Abbreviations: chr, chromosome; CNG, copy number gain; CNL, copy number loss; hFF, human foreskin fibroblast; hGF, human gingival fibroblast; iPSC, induced pluripotent stem cell.
Genetic stability of the human iPSCs derived on poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH)-grafted plates (GPs). The genomic stability of the three human iPSC lines derived on PMEDSAH-GPs was tested 9 months after derivation and continuous in vitro culture. (A): Representative standard G-banding metaphase karyotyping of one of the three human iPSCs derived on PMEDSAH-GPs showing normal male karyotype. (B): Ideogram summarizing chromosome losses and gains (left and right, respectively) of the three human iPSCs as detected by high-resolution array-based comparative genomic hybridization. No mutations are localized in chromosome loci where genes related to stem cells, cancer, or culture adaptation are localized. Abbreviations: chr, chromosome; CNG, copy number gain; CNL, copy number loss; hFF, human foreskin fibroblast; hGF, human gingival fibroblast; iPSC, induced pluripotent stem cell.

This paper demonstrates that it is possible to generate transgene-free, stable iPSCs on a synthetic substrate.  This type of platform has the potential to meet the good manufacturing practices that must be used to make products for clinical use.

First Stem Cell Therapy Recommended for Approval in European Union


The EMA, which is short for the European Medicines Agency, has recommended approval for a treatment called Holoclar.  Holoclar is the first therapy product that contains stem cells to be recommended for approval in the European Union (EU). Holoclar is being marketed as a treatment for moderate to severe limbal stem cell deficiency (LSCD) due to physical or chemical burns to the eye in adults. In fact, Holoclar is the first medicine recommended for LSCD, a condition that can result in blindness.

Holoclar can be transplanted into the eye after removal of the corneal epithelium (the outer layer of the cornea). Holoclar is made from a biopsy taken from a small, undamaged area of the patient’s cornea. These limbal stem cells are then grown in the laboratory using cell culture techniques. Holoclar is a potential alternative to transplantation for replacing altered corneal epithelium. Clnical trials with Holoclar have been shown to increase the chances of a successful corneal transplant where the injury has caused extensive eye damage. Holoclar is produced by Chiesi, a pharmaceutical company based in Parma, Italy.

The recommendation to approve Holoclar was made by the EMA’s Committee for Medicinal Products for Human Use (CHMP). CHMP made their recommendation on basis of the benefits of Holoclar, which are its ability to repair the damaged ocular surface, to improve or resolve symptoms of pain, photophobia and burning and to improve the patient’s visual acuity. This assessment was the work of the Committee for Advanced Therapies (CAT). The approved indication for Holoclar is: “Treatment of adult patients with moderate to severe limbal stem cell deficiency (defined by the presence of superficial corneal neovascularisation in at least two corneal quadrants, with central corneal involvement, and severely impaired visual acuity), unilateral or bilateral, due to physical or chemical ocular burns. A minimum of 1-2 square millimeters of undamaged limbus is required for biopsy.” CAT and CHMP considered that Holoclar provided a first treatment option for LSCD and recommended a conditional marketing authorization. The authorization is conditional because the clinical data available for Holoclar is based on studies that are ongoing as treated patients are watched after their eye surgery. This the data collection is not yet comprehensive, and additional study on the use of Holoclar needs to be conducted.

The opinion adopted by the CHMP at its December 2014 meeting is an intermediary step on Holoclar’s path to patient access. The CHMP opinion will now be sent to the European Commission for a decision on an EU-wide marketing authorization.

Discovery of New Stem Cell Class Might Accelerate Research


An international team of scientists has discovered a new class of stem cell. This project consisted of a massive collaboration between over 50 scientists on four continents, that has been affectionately named, “Project Grandiose.” This new class of stem cells, known as a F-class cell, opens new and exciting avenues for generating designer cells that could be safer and more efficiently used in therapy.

Andras Nagy, Ph.D., from the University of Toronto’s Institute of Medical Sciences led this group in conducting a high-resolution characterization of the molecular events that are required for the reprogramming of stem cells. In particular, Nagy and his colleagues were interested in ways to control the path to pluripotency. In this analysis, they discovered an alternative reprogrammed cell, which they called F-class stem cells.

It has been known for many years that when mature, adult cells are reprogrammed into induced pluripotent stem cells (iPSCs) by means forcing expression of key transcription factors (Oct4, Klf4, Sox2, and c-Myc), some cells will stably not express the pluripotency gene Nanog, and fail to acquire full pluripotency, even though these cells look like embryonic stem cells (see Fussner, E. et al. EMBO J. 30, 1778–1789 (2011); Sridharan, R. et al. Cell 136, 364–377 (2009); and Chen, J. et al. Nature Genet. 45, 34–42 (2013)). These partially reprogrammed cells seem to indicate that there are other cell types that can be formed by reprogramming that are not fully pluripotent. Strangely, some labs have reported that treating partially pluripotent cells with vitamin C can reprogram to cells to full pluripotency (Esteban, M. A. et al. Cell Stem Cell 6, 71–79 (2009)).

Nagy and his colleagues used a whole battery of tests to take detailed snapshots of every stage of reprogramming, and in the process, revealed an alternative state of pluripotency. They discovered that high levels of expression of the four reprogramming factors generates cells that do not form typical ESC-like colonies in culture, but are still pluripotent. These are the F-type cells.  F-type cells derived their name from the fuzzy boundaries they form when they grow in culture.

When F-type cells were compared to embryonic-like stem cells, the F-type cells are easier to make, less expensive, and faster to grow. Thus F-class stem cells can be produced more economically in large quantities and this should accelerate drug-screening efforts, disease modeling, and eventually the development of treatments for different illnesses.