Bioengineered Trachea Implanted into a Child

Hannah Genevieve Warren was born in 2010 in Seoul, South Korea with tracheal agenesis, which is to say that she was born without a trachea. Hannah had a tube inserted through her esophagus to her lungs that allowed her to breathe. Children with tracheal agenesis usually die in early childhood, 100% of the time. No child with this condition has ever lived past six years of life. Hannah spent the first two years of her life at the Seoul National Hospital before she was transported to Illinois for an unusual surgery.

While at the Children’s Hospital of Illinois, on April 9, 2013, Hannah had a bioengineered trachea transplanted into her body. This trachea was the result of a remarkable feat of technology called the InBreath tracheal scaffold and bioreactor system that was designed and manufactured by Harvard Bioscience, Inc. Harvard Bioscience, or HBIO, is a global developer, manufacturer and marketer of a broad range of specialized products, primarily apparatus and scientific instruments, used to advance life science research and regenerative medicine.

InBreath tracheal scaffold
InBreath tracheal scaffold

Hannah’s tracheal transplant was the first regenerated trachea transplant surgery that used a biomaterial scaffold that manufactured by the Harvard Apparatus Regenerative Technology (HART) Inc., a wholly owned subsidiary of Harvard Bioscience. HART ensured that the scaffold and bioreactor were custom-made to Hannah’s dimensions. Then the scaffold was seeded with bone marrow cells taken from Hannah’s bone marrow, and the cells were incubated in the bioreactor for two days prior to implantation. Because Hannah’s own cells were used, her body accepted the transplant without the need for immunosuppressive (anti-rejection) drugs.

InBreath Bioreactor
InBreath Bioreactor

The surgeons who participated in this landmark transplant were led by Dr. Paolo Macchiarini of Karolinska University Hospital and Karolinska Institutet in Huddinge, Stockholm and Drs. Mark J. Holterman and Richard Pearl both of Children’s Hospital of Illinois. This surgery was approved by the FDA under an Investigational New Drug (IND) application submitted by Dr. Holterman.

Dr. Mark Holterman, Professor of Surgery and Pediatrics at University of Illinois College of Medicine at Peoria, commented: “The success of this pediatric tracheal implantation would have been impossible without the Harvard Bioscience contribution. Their team of engineers applied their talent and experience to solve the difficult technical challenge of applying regenerative medicine principles in a small child.”

David Green, President of Harvard Bioscience, said: “We would like to congratulate Dr. Macchiarini, Dr. Holterman, Dr. Pearl and their colleagues for accomplishing the world’s first transplant of a regenerated trachea in a child using a synthetic scaffold and giving Hannah a chance at a normal life. We also wish Hannah a full recovery and extend our best wishes to her family.”

Hannah’s surgery is the seventh successful implant of a regenerated trachea in a human using HART technology. Prior successes included the first ever successful regenerated trachea transplant in 2008, the first successful regenerated trachea transplant using a synthetic scaffold in 2011, and the commencement of the first clinical trial of regenerated tracheas in 2012. HART has plans to commence discussions with the FDA and EU regulatory authorities in the near future regarding the clinical pathway necessary to bring this new therapeutic approach to a wider range of patients who are in need of a trachea transplant.

Stem Cell Treatments for Large Ankle Cartilage Lesions

The Regenexx blog has called attention to to a study that shows that older patients with ankle problems can benefit from mesenchymal stem cell treatments of the ankle.

The paper referenced by Centeno on his blog was published in the American Journal of Sports Medicine 2013 May;41(5):1090-9, and is entitled, “Clinical outcomes of mesenchymal stem cell injection with arthroscopic treatment in older patients with osteochondral lesions of the talus.” The authors of this paper are Kim YS, Park EH, Kim YC, Koh YG. who all hale from the Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul, South Korea.

The paper notes that there are no generally accepted, ideal treatments for “osteochondral lesions of the talus”, which is in plain English means cartilage erosions of the large bone in the foot that articulates with the base of the lower leg bones. That bone, the talus, sits above the heel bone (calcaneus) and is covered with articular cartilage to absorb shocks that occur between blows from the fibula and tibia and the talus. Over time, wear and tear can erode this cartilage and the best way to go about fixing these osteochondral lesions of the talus or OLTs as they are called, is not at all clear. Centeno mentions in his blog that he and his colleagues have been treating OLTs with mesenchymal stem cells for some time (since 2006). In this paper, Kim and others tested the ability of bone marrow mesenchymal stem cells (MSCs) to provide relief from OLT.


Kim and colleagues compared the outcomes of patients who had received MSC injections and arthroscopic marrow stimulation treatments against the outcomes of those patients who had received only arthroscopic marrow stimulation treatment alone.

In this study, from a starting group of 107 patients with OLTs that were treated arthroscopically, only those patients older than 50 years (65 patients) were included in this study. Kim and others divided the patients into 2 groups: 35 patients (37 ankles) were treated with arthroscopic marrow stimulation treatment alone (group A) and 30 patients (31 ankles) who underwent MSC injection along with arthroscopic marrow stimulation treatment (group B).

The clinical outcomes for these patients were assessed according to the visual analog scale (VAS) for pain, but there were other measurements as well. For example, how active were the patients? A Tegner score determined how active patients were, which is important because the more active a patient is, the less likely they are to be in pain. Other assessments included the American Orthopaedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Scale, which measures how well you walk and how much pain you experience when you do it, and other scores for the foot and how well it works

The outcomes for the study were as follows. Both groups showed a decrease in pain. Group A had a VAS score that started at 7.2 ± 1.1 and fell to 4.0 ± 0.7, whereas group B started at 7.1 ± 1.0 and decreased to 3.2 ± 0.9. Therefore the patients in group B showed a slightly greater decrease in pain over the non-MSC group.

As for the AOFAS score in each group, which measures ankle function and pain while using the ankle, again, both groups showed improvement. Patients in group A went from 68.0 ± 5.5 to 77.2 ± 4.8, and patients in group B went from 68.1 ± 5.6 to 82.6 ± 6.4. Thus the patients who received the MSC treatments used their ankles better than those patient who did not receive MSC treatments, and they also used their ankles with less pain.

A different measure of ankle function, the Roles and Maudsley score, also increased significantly in group A patients as opposed to group A patients at postsurgical follow up (1-4 years after the surgery). However, the real “money” finding of this research was that the patients who had received the MSC injections were significantly more active after surgery than the non-MSC patients. The activity score in patients from group A. Group A patients had a Tegner activity score that went from 3.5 ± 0.8 to 3.6 ± 0.6. That is not a significant increase. However the patients in group B, who had received the injections of their own MSCs into the ankle, had Tegner activity scores that improved from 3.5 ± 0.7 to 3.8 ± 0.7 (P = .041). Thus the patients who had received the MSC injections had less pain, better ankle function with less pain, better ankle and foot function 1-4 years after surgery, and were significantly more active after surgery.

From these data, the authors concluded that injection of MSCs with marrow stimulation treatment was a treatment option in patients older than 50 years than marrow stimulation treatment alone. THis was especially the case if the OTL was larger than 109 square millimeters. Also, those patients in group A who had subchondral cysts did not fare well with their treatment, but there was no such correlation between a poor clinical outcome and the presence of a subchondral cyst in patients from group B.

subchondral cyst in the ankle
subchondral cyst in the ankle

Therefore, even though MSC treatments for OTLs is still in its early stages of development, they seem to have the potential to treat of OLTs in patients older than 50 years. However, this study, while not tiny, is still not all that large, and larger studies are warranted.

A Model System for A Devastating Childhood Disease

A Japanese research team from Fukuoka, Japan, specifically from the Department of Pediatrics at the University of Fukuoka, Japan, have used induced pluripotent stem cell technology to make neurons from human patients who suffer from a rare, devastating condition known as Dravet syndrome as a model system.

Dravet syndrome (DS) causes difficult to control seizures within the first year or two of life and later causes cognitive deficits and autistic traits. Dravet’s syndrome is caused by genetic alterations in the SCN1A gene, which encodes the α-subunit of the voltage-gated sodium channel.

DS is very rare – 1/30,000 children, but the mutation is typically not inherited from either parent, but occurs spontaneously in the baby’s cells during development. The best model systems to date are genetically engineered mice, but the differences between human and mouse brains limits the usefulness of this model system.

To make a better model system, workers from the laboratory of Shinichi Hirose took skin biopsy samples from a DS patient, and converted those skin cells into induced pluripotent stem cells (iPSCs), which were then differentiated into neurons. In particular, the neurons that malfunction in DS patients are GABAminergic neurons, and by differentiating DS iPSCs into GABAminergic neurons, Hirose’s laboratory made a model system for DS patients that could be grown in a laboratory culture dish.

Hirose explained their results this way: “From research in mice we believed that SCN1A mutations affect GABAminergic neurons in the forebrain from signaling properly. From the human neurons we also found that GABAminergic neurons were affected by DS, especially during intense stimulation. These patient-specific cell provide an unparalleled insight into the mechanism behind DS and a unique platform for drug development.”

Perhaps such experiments could eventually lead to regenerative treatments for DS patients as well.

Turning Adult Cells into Early Stage Neurons and Bypassing the Pluripotent Cell Stage

Researchers at the University of Wisconsin, Madison have converted skin cells from monkeys and humans into early neural stem cells that can form a wide variety of nervous system-specific cells. This reprogramming did not require converting adult cells into induced pluripotent stem cells or iPSCs. Su-Chun Zhang, professor of neuroscience and neurology at the University of Wisconsin, Madison, served as the senior author of this research. Bypassing the ultraflexible iPSC stage was the key advantage in this research, accord to Zhang.

Zhang added, “IPSC cells [sic] can generate any cell type , which could be a problem for cell-based therapy to repair damage due to disease in the nervous system.” In particular, the absence of iPSCs greatly reduces the risk of tumor formation in the recipient of the stem cell therapy.

There is a second advantage to this procedure. Namely that iPSC generation usually requires the recombinant viruses that deliver genes to the adult cells. These viruses, retroviruses, insert their genes directly into the genomes of the host cell. While there are ways are using such viruses, the use of retroviruses is definitely the most popular strategy for converting adult cells into iPSCs.

Retroviral life cycle
Retroviral life cycle

However, the procedure used in Zhang’s laboratory, utilized recombinant Sendai viruses that do not integrate their genes into the genome of the host cell, but expressed them transiently, after which, the exogenous genes are degraded.

Sendai virus
Sendai virus

Jaingfeng Lu, a postdoctoral researcher in Zhang’s lab, removed skin cells from monkeys and people, and exposed them to recombinant Sendai viruses that contained the four genes normally used to make iPSCs for 24 hours. Then Lu heated the cells to thirty-nine degrees to kill the viruses and prevent the cells from becoming iPSCs. However, 13 days later, Lu found that the cells had become induced neural progenitors or iNPs. When implanted into newborn mice, the iNPs grew normally and differentiated into neural cell types without forming any tumors.

While other researchers have managed to convert adult cells directly into neurons, Zhang admitted that he had a different goal. “our idea was to turn skin cells into neural progenitors, cells that can produce cells relating to the neural tissue. These progenitors can be propagated in large numbers.”

the research overcomes limitations of previous efforts, according to Zhang. The Sendai, which produces little more than a cold, is not a severe pathogen, does not integrate its genes into the genome of the host cell, does not cause tumors, and is considered safe, since it can be killed by heat within 24 hours. This illustrates how fevers in our bodies can kill off cold viruses. Secondly, the iNPs have a greater ability to grow in culture. Third, iNPs are far enough along in their differentiation so that they can only form nervous system-specific cell types. They cannot form muscle or live. However, the iNPs can form many more specialized cells.

Interestingly, the neurons produced from the iNPs had the characteristics of neurons normally formed in the back part of the brain, something that is potentially helpful. As Zhang noted, “For therapeutic use, it is essential to use specific types of neural progenitors. We need region-specific and function-specific neuronal types for specific neurological diseases.”

Progenitor cells grown from the skin of ALS or spinal muscular atrophy patients can be used to make a whole host of neural cells in order to model each disease and allow rapid drug screening. Such cells could also be used to treat patients with neurological disease too.

“These transplantation experiments confirmed that the reprogrammed cells indeed belong to ells of the intended brain regions and the progenitors produced the three major classes of neural cells: neurons, astrocytes, and oligodendrocytes. This proof-of-principle study highlights the possibility to generate [sic] many specialized neural progenitors for specific neurological disorders.”

Neural progenitors
Neural progenitors

Lu, Jianfeng, Liu, Huisheng, Huang, Cindy Tzu-Ling, Chen, Hong, Du, Zhongwei, Liu, Yan, Sherafat, Mohammad Amin, Zhang, Su-Chun.  Generation of Integration-free and Region-Specific Neural Progenitors from Primate Fibroblasts.  2013/05/02. Cell Reports 2211-1247.

Wesley Smith and Cloning

My favorite bioethicist, Wesley Smith said this about human cloning in his prescient book: A Consumer’s Guide to A Brave New World:

We can pursue biotechnology to treat disease and improve the human condition, while retaining sufficient humility and self-restraint to keep ourselves from endangering the intrinsic value of human life. Or, we can hubristically rush onto the very anti-human path warned against by Aldous Huxley, driven by our thirst for knowledge, vast profits, and obsession with control and vastly expanded life spans.

These issues are too important to be “left to the scientists.” Nor can we afford to allow the marketplace to determine what is right and what is wrong. The stakes are too high, the potential impact on each and every one of us too profound, to remain passive and indifferent to the decisions that are to be made. It is our duty to participate in the crucial cultural and democratic debates over biotechnology. The human future, quite literally, depends on it.

Prophetic and poignant – and DEAD RIGHT!!

The Archbishop of Denver Speaks Out: Cloning Kills the Smallest Among Us and the Next Victims WIll be Us

Samuel Aquila is archbishop of the Archdiocese of Denver, Colorado and has weighed in with regards to the cloning of human embryos. I am not a Roman Catholic, and Fr. Aquila is not a person whose religious authority I am obligated to accept de fide. Nevertheless, his stance on this subject is reasoned and was published on the National Review Online website here. It is well worth reading.

Human Stem Cells From Cloned Embryos: What Horrors WIll Follow?

First the news, then the commentary. Here’s the news:

In the May 14th edition of the international journal Cell, Shoukhrat Miltalipov from the Oregon Health and Science University, reported the derivation of human embryonic stem cells from cloned human embryos. This is the first time this has been successfully reported. In 2004, a South Korean researcher, Woo Suk Hwang, reported that his laboratory had succeeded in making patient-specific human embryonic stem cells from cloned embryos, but his papers were later shown to be completely fraudulent, and Hwang, in a word, walked. For more on this sad, sordid event, see my “Catastrophic Cloning Caper” here.

Many laboratories have tried and failed to get cloned human embryos to live long enough to get embryonic stem cells from them. The cloning procedure produces a very abnormal embryo that dies very early during development.

How did Mitalipov succeed when so many others before him had failed? Mitalipov honed his cloning protocol in work with early embryos from Rhesus macaques, and during this work, Mitalipov and his coworkers discovered that including caffeine with the mix of chemicals used during donor removal and transplantation into the host egg prevents the oocytes that have just had their nuclei removed from dividing prematurely, and if these oocytes are used in a cloning experiment, they survive longer than oocytes treated with standard cloning techniques.

“It was a huge battery of changes to the protocols over a number of different steps,” said Mitalipov. “I was worried that we might need a couple of thousand eggs to make all these optimizations, to find that winning combination.”

The procedure used in this paper, cloning, is more technically known as “somatic cell nuclear transfer” or SCNT. SCNT requires human eggs that are extracted from female volunteers of reproductive age who are given several drugs to hyperstimulate their ovaries, which then ovulate several eggs at a time. The eggs are harvested by means as aspiration, and used in SCNT.

For SCNT, the egg nucleus is removed by means of a micropipette. The egg is ever so gently squeezed until the nucleus, which is usually off to one side in the egg, protrude through the cell membrane, and the nucleus is sucked off with the micropipette. Then a body cell; in this paper, fibroblasts from the skin were used, is laid next to the nucleus-less egg, and an electric current is pulsed through the two cells, which causes them to fuse. This fusion converts the egg, which used to have one set of every chromosome, into a cell that now has two sets of every chromosome, and the egg cell, begins to divide and recapitulate the events of early development. This is also referred to as cloning.


Sperm and eggs have chromosomes that have been modified in specific ways. When the sperm and egg fuse, the process of fertilization begins, and the modifications to the chromosomes serve their purpose during the early stages of development, but those modifications and gradually undone as development proceeds. This phenomenon is known as genetic imprinting and it is very common in mammals. For a good paper on genetic imprinting see Wood AJ, Oakey RJ (2006) Genomic Imprinting in Mammals: Emerging Themes and Established Theories. PLoS Genet 2(11): e147. doi:10.1371/journal.pgen.0020147.

Since cloned embryos have a genome that is not properly imprinted, its development is hamstrung to one degree or another. Most researchers were unable to get cloned human embryos to survive past the 8-cell stage. However, by including caffeine in the SCNT medium during egg nucleus removal and transplantation of the donor nucleus into the host egg, enough of the cloned embryos survived to the 150-cell blastocyst stage to allow for the derivation of embryonic stem cells. Even though SCNT is an exceedingly inefficient process, Mitalipov was able to derive six embryonic stem cells lines from 128 eggs, which is about a 4% success rate.

George Daley of Boston Children’s Hospital and the Harvard Stem Cell Institute, who was not involved in the research, said of it: ““I think it is a beautiful piece of work.” He continued: “This group has become remarkably proficient at a very technically demanding procedure and [has] shown that SCNT-ESCs may in fact be a practical source of cells for regenerative medicine.”

Mitalipov and his group analyzed four of the cloned embryonic stem cell lines and found that their NT-hESCs could successfully differentiate into beating heart cells in culture dishes. Also, they could differentiate into a variety of cell types in teratoma tumors when transplanted into live, immunocompromised mice. These stem cells also had no chromosomal abnormalities, and displayed fewer problematic epigenetic leftovers from parental somatic cells than are typically seen in induced pluripotent stem cells (although, for the life of me, no one has shown that these epigenetic holdovers are a big problem for regenerative medicine). Mitalipov said more comparisons are required, however.

“We are now left to analyze the detailed molecular nature of SCNT-ES cells to determine how closely they resemble embryo-derived ES cells and whether they have any advantages over iPS cells,” added Daley. “iPS cells are easier to produce and have wide applications in research and regenerative medicine, and it remains to be shown whether SCNT-ES cells have any advantages.”

Mitalipov, however, did point out one fundamental difference between NT-ESCs and iPSCs: their nuclear genomes come from the donor cell, but NT-hESCs contain mitochondrial DNA (mtDNA) from the host egg cell. Therefore, SCNT reprograms the cell but also corrects any mtDNA mutations that the donor may carry. Thus, patient-specific NT-hESCs could be used to treat people with diseases caused by mitochondrial mutations. “That’s one of the clear advantages with SCNT,” Milatipov said.

The cells used for this cloning experiment came from infants.  It still remains for cloning to succeed with adult cells as the donor cells.

Now for the commentary:

Regular readers of this blog will already know that I am deeply opposed to human cloning in any form.  It is the equivalent of making people for spare parts.  This is immoral and barbaric.  I predicted some time ago (OK not so long ago, 4 years to be exact), that the technical problems with human cloning would be solved and scientists would one day clone a human embryo.  Now that it is here, I hope that people are as horrified by it as I am.

“Get over it.  It’s an embryo and a cloned one at that.” you might say.  But what if the malady that doctors want to cure is poorly served by embryonic stem cells made from cloned embryos and a cloned fetus is a better source of cells?  Do we allow gestation of the cloned embryo to the fetal stage so that we can dismember it and take its tissue?  Let’s bring this home.  What if the cells needed to come from a five-year old?  Can we justify that because the kid was cloned?

“But wait, that’s a five-year old and this is an embryo,” you say.  But you were once a blastocyst.  You did not pass through the blastocyst stage, you WERE a blastocyst.  The only difference between the blastocyst and you now is time, environment, degree of dependence, and size.  Are any of these differences morally significant when it comes to whether or not we can kill you?  Can we kill all the short people?  Can we kill all the younger people because they are not as well-developed?  Can we kill people who are dependent on others (that includes everyone mate, so put your hand down)?  Can we kill those in a different location (genocide anyone)?  None of these categories constitutes a good reason for terminating someone’s life.  Likewise, none of these changes renders you essentially different from who and what you are.  To kill someone at the earliest stages for their tissue is simple murder, and we use size, location, extent of development, location and degree of dependence to salve of consciences for doing it, but that won’t define what we are doing.

People will go on and on about the great advances that could lead to.  Sorry, I’m not buying that one.  Embryonic stem cells have been promising that one for the last 15 years with pert near little to nothing to show for it.  This discovery is a great technical advance, but it opens to door to reproductive cloning – an even bigger mistake, and fetus farming, in which we destroy our own children in the womb, not because they are in inconvenience to us, but because we want their tissues to save our lives.  Now children, rather than being a blessing, are merely tissues to be harvested.  We have become like the Greek gods from the stories of old who ate their own children.  May God forgive us.