Chicken Induced Plurpotent Stem Cells Made With Minicircles

The safety of induced pluripotent stem cells (iPSCs) haws been debated in several studies and publications.  Original studies of the genetic differences between the cellular sources of iPSCs and the iPSCs derived from them tended to show a whole gaggle of new mutations that seemed to not appear in the original cells.  Therefore, several commentators warned about the “dark side of pluripotency.”. However, other studies that utilized higher-resolution techniques showers that many of these mutations that occurred in iPSCs did exist in the original cells before their reprogramming, but that these mutations occurred at low frequencies, but became amplified during the culturing of reprogrammed cells.

One feature that has received less attention in these discussions of the safety of iPSC derivation is that the method by which iPSCs are made has distinct consequences for the stem cells that are made.  Typically, methods that utilize gene vectors that do not integrate into the genomes of the host cells are inherently safer than those vectors that do integrate.  PiggyBac transposon vectors integrate, but self-excise soon after their integration, and, therefore, do not leave a trace or their previous integration.  Minicircles also do not integrate and tend to produce safer iPSCs.  For this reason, this present paper is of interest to us.

Franklin West and his colleagues at the University of Georgia have made chicken iPSCs using minicircles to reprogram adult cells.  West was interested in using iPSCs to make recombinant chickens, since chickens are a rather primary food source and major component of economic development in several countries.  Making transgenic or recombinant chickens by means of stem cell technology makes it possible to make animals with improved meat and egg production or disease resistance.

To this end, West and his group made chicken (c) iPSCs from skin fibroblast cells by means of a nonviral minicircle reprogramming method.  This resulted in ciPSCs that showed excellent stem cell appearance and expressed key stem cell marker genes (alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1).  These cells also showed very rapid growth in culture and expressed high levels of the enzyme telomerase, which is an enzyme that is vital for the maintenance of chromosomes.

When West and his research group transplanted late-passage ciPSCs into stage X chicken embryos, the cIPSCs successfully integrated into the growing embryo and contributed to tissues derived from all three primary germ layers (ectoderm, mesoderm, and endoderm).  These ciPSCs also contributed to the gonads, which means that the ciPSCs could make gametes that could contribute to the production of a new generation of chicken.

These ciPSCs provide an exciting new tool to create transgenic chickens and has broad and exciting implications for agricultural and transgenic animal fields at large.  However, it also demonstrates that iPSCs can be safely produced and used for agricultural purposes.  This means that if non-integration-based or non-viral-based techniques are used to make iPSCs it should be possible to make them safely for therapeutic purposes also.

Kyoto University Scientist Plans iPSC Clinical Trial for Parkinson’s Disease Patients

According to the Japan Times, Kyoto University’s Jun Takahashi and his team have plans to launch a clinical study for Parkinson’ disease patients that will utilize cells derived from induced pluripotent stem cells made from the patient’s own cells.

In an interview with Takahashi, the Japan Times reported on Wednesday of this week that he hopes to develop the induced Pluripotent Stem Cell (iPSCs) treatment as soon as possible so that Kyoto University Hospital can provide this treatment by fiscal year 2018 as a designated advanced medical technique that can be used in combination with other conventional treatments and medicines already covered by various insurance policies. Takahashi also expressed his hope that by fiscal year 2023, public health insurance will pay for his treatment.

For this clinical study, Parkinson’s disease patients whose conditions have progressed to the point where their medications are no longer effective will be the primary targeted group.  “It will take a long time” to establish an effective treatment for the progressive disorder, which is incurable at present, Takahashi said, stressing the importance of maintaining a positive attitude toward development and not losing hope.

Parkinson’s disease causes the nerve cells in the brain that utilize the neurotransmitter dopamine to die off.  The death of these dopaminergic neurons adversely affects voluntary muscle movement.

The design of this clinical study will include the production of iPSCs from adult cells collected from participating patients.  These stem cells will be differentiated into neural stem cells that make dopaminergic neurons.  These dopaminergic neuron precursor cells will be transplanted back into the midbrains of the donors before they develop into nerve cells, according to Takahashi.  This way, all injected cells will still have the capacity to divide and migrate once implanted into the brain, but they will still have the capacity to form dopaminergic neurons.

Takahashi’s team will also seek to develop a method for producing a nerve cell drug created from cells taken out of healthy people, to ease the financial burden on patients, he said, since the derivation of iPSCs remains prohibitively expensive.

Takahashi also said he aims to being clinical trials by March 2019.

Regenerating Tooth Roots With Biomaterials

Several different types of stem cells can regenerate tooth enamel, but regenerating the tooth root has proven quite difficult.


As you can see from the image above, the tooth root is covered with a tough, fibrous covering called the cementum.  The cementum connects the tooth root to the alveolar bone of the upper and low jaw by means of the periodontal membrane.  the cementum is a thin layer of bone-like material that covers the roots.  It is yellowish and softer than either dentine or enamel.  It is made by a layer of cementum-producing cells called cementoblasts that are adjacent to the dentine.  The periodontal ligament is cellular and its fibers hold the tooth in its socket, which are embedded in the cementum, as shown in the micrograph below.  The complexity of this structure shows you why regenerating this structure is so difficult.

Cementum-peridontal ligament

Howwever, a new study from the laboratory of Weihua Guo at Sichuan University, China has shown that platelet-rich fibrin (PRF) and treated dentin matrix (TDM) can concentrate a variety of various growth factors that summon native stem cells to them, and induce them to regenerate the tooth root.

Guo’s laboratory examined the ability of PRF and TDM to summon endogenous stem cells to the site of an extracted tooth in order to initiate regeneration of the tooth root.  Tooth roots contain soft and hard periodontal tissues, and if periodontal ligament stem cells (PDLSCs) and bone marrow mesenchymal stem cells (BMSCs) could be recruited to the site of tooth extraction by PRF and TDM, then maybe they could initiate tooth root regeneration.

Beagles were used as a transplantation model for this experiment.  After tooth extraction PRF and TDM implants were embedded in the tooth socket.  Also, these matrices were examined in cell culture with  PDLSCs and BMSCs.

PRF significantly recruited and stimulated the growth of both PDLSCs and BMSCs in culture.  In combination, PRF and TDM induced cell differentiation of these implanted stem cell populations.  PRF and TDM induced the expression of mineralization-related genes, such as bone sialoprotein (BSP) and osteopotin (OPN) after only one week in culture.

When implanted into the tooth sockets of beagles that had teeth extracted, transplantation platelet-rich fibrin made from the dog’s own blood products, and TDM made from other animals into fresh tooth extraction socket successfully regenerated the tooth root 3 months after the surgery.  The cementum and periodontal ligament (PDL)-like tissues with properly orientated fibers were clearly present, and the presence of these structures is indicative of functional restoration.

These results suggest that tooth root and the connection of the tooth root to the alveolar bone by cementum and peridontal ligaments can be effectively regenerated through the implantation of PRF and TDM in a tooth socket.  It seems to achieve this regeneration by summoning BMSCs and PDLSCs.  These cues provided by these matrices and the microenvironment provided by the tooth socket are key factors for this regeneration. This strategy provides a genuine clinical pathway toward tooth root regeneration in human patients with destroying human embryos.

A Genetic Recipe To Convert Stem Cells into Blood

University of Wisconsin at Madison Stem Cell researchers led by Igor Slukvin discovered two genetic programs that can convert pluripotent stem cells into the wide array of white and red blood cells found in human blood (pluripotent means “capable of developing into more than one organ or tissue and not fixed as to potential development).

This research has ferreted out the actual pathway used by the developing human body to make blood-based cells at the early stages of development.

During embryonic development, blood formation, which includes the formation of blood cells and blood vessels from the same progenitor cell; a cell called a hemangioblast. This begins in week three of development in the extraembryonic mesoderm or the primary embryonic umbilical sac, which is also known as the yolk sac. Also, the connecting stalk and chorion contain blood islands as well. These blood islands are rich in particular growth factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PIGF). The blood islands form clusters with two cell populations; peripheral cells (angioblasts) that form the endothelial cells that form vessels. These networks of vessels extend and fuse together to form a robust a network. The cores of the blood islands (hemocytoblasts) form blood cells. Initially all vessels (arteries and veins) look the same. Blood formation occurs later in week 5, and occurs throughout the embryonic mesenchyme (connective tissue), and then moves to the liver, and then the spleen, and then bone marrow.

Embryonic red blood cells
Embryonic red blood cells

Hematopoietic stem cells (HSCs), the stem cells that form the blood cells, form from the wall of the aorta, which is the major blood vessel in the embryo. In the aortic wall, cells called hemogenic endothelial cells bud off progenitor cells that become HSCs.

A course of transcription factors have now been identified by Slukvin and his team as the triggers that switch these cells into HSCs. Two groups of transcriptional regulators can induce distinct developmental programs from pluripotent stem cells. The first developmental program, directed by the transcription factors ETV2 and ​GATA2, the pan-myeloid pathway, switches cells into the myeloid lineage (the myeloid lineage includes red blood cells, platelets, neutrophils, macrophages, basophils and eosinophils). The second developmental pathway, directed by the transcription factors GATA2 and ​TAL1, directs cells into the erythro-megakaryocytic pathway. In either cases, these transcription factors directly convert human pluripotent stem cells into an endothelium, which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential.


In Slukvin’s laboratory, treatment of either ETV2 and ​GATA2 or GATA2 and ​TAL1 induced cells to make the complete range of human blood cells. Slukvin said of these experiments, “This is the first demonstration of the production of different kinds of cells from human pluripotent stem cells using transcription factors.” Transcription factors bind to DNA at specific sites and regulate gene expression.

Slukvin continued: “By overexpressing just two transcription factors, we can, in the laboratory dish, reproduce the sequence of events we see in the embryo.”

Slukvin and his co-workers showed that his technique produced blood cells by the millions. For every million stem cells, it was possible to produce 30 million blood cells.

Slukvin and his colleagues did not use viruses to genetically modify these stem cells. Instead they used modified RNA to induce overexpression of these transcription factors. Such a technique avoids genetic modification of cells and is inherently safer.

“You can do it without a virus, and genome integrity is not affected,” said Slukvin.  This technique might also work to differentiate pluripotent stem cells into other cell types, such as pancreatic beta cells, brain-specific cells, or liver cells.

Despite these successes, Slukvin says that the “Holy grail” of hematopoietic research is to differentiate pluripotent stem cells into HSCs.  Since HSC transplants are used to treat multiple myeloma and other types of blood-based cancers as well, making HSCs in the laboratory remains a significant goal and challenge as well.

“We still don’t know how to do that,” said Slukin, “but our new approach to making blood cells will give us an opportunity to model their development in a dish and identify novel hematopoietic stem cell factors.”

High-Dose Stem Cell Treatments in Chronic Heart Patients Increases Survival Rates

The DanCell clinical trial was conducted about seven years ago at the Odense University Hospital, Odense, Denmark by a clinical research team led by Axel Diederichsen. The DanCell study examined 32 patients with severe ischemic heart failure who had received two rounds of bone marrow stem cell treatments.

The DanCell study was small and uncontrolled. However, because the vast majority of stem cell-based clinical trials have examined the efficacy of stem cell treatments in patients who have recently experienced a heart attack, this study was one of the few that examined patients with chronic heart failure.

In this study, patients had an average ejection fraction of 33 ± 9%, which is in the cellar – normal ejection fractions in healthy patients are in the 50s-60s. Therefore, these are patients with distinctly “bad tickers.” All 32 patients received two repeated infusions (4 months apart) of their own bone marrow stem cells, but these stem cell infusions were quantitated to determine the number of “CD34+” cells and the number of “CD133+” cells. CD34 is a cell surface protein found on bone marrow hematopoietic stem cells, but it by no means exclusive to HSCs. CD133 is also a cell surface protein found, although not exclusively, on the surfaces of cells that form blood vessels and blood vessels cells as well.

Initially, patients showed no improvements in heart function after 12 months. However, when patients were classified according to those who received the most or the least number of CD34+ cells, a curious thing emerged: those who received more CD34+ cells had a better chance of surviving than those who received fewer CD34+ cells.

Is this a fluke? To determine if it was, Diederichsen and his colleagues followed these patients for 7 years after the bone marrow infusion. When Diederichsen and his colleague recorded the number of deaths and compared them with the number of CD34+ cells infused, the pattern once again held true. The CD34+ cell count and CD133+ cell count did not significantly correlate with survival, but the CD34+ cell count alone was significantly associated with survival. In the authors own words: “decreasing the injected CD34 cell count by 10[6] increases the mortality risk by 10%.”

The conclusions of this small and admittedly uncontrolled study: “patients might benefit from intracoronary stem cell injections in terms of long-term clinical outcome.”

Three things to consider: Patients with heart conditions have poorer quality bone marrow stem cell numbers. Therefore, allogeneic stem cells might be a better way to go with this patient group. Secondly, the Danish group used Lymphoprep to prepare their bone marrow stem cells, which has been used in other failed studies, and the stem cell quality was almost certainly an issue in these cases (see the heart chapter in my book The Stem Cell Epistles for more information). Therefore, independent tests of the bone marrow quality are probably necessary as well or a different isolation technique in general. Also, a controlled trial must be run in order to confirm the efficacy of bone marrow stem cell infusions for patients with chronic ischemic heart disease. Until them, all we can conclude is that intracoronary injections of a high number of CD34+ cells may have a beneficial effect on chronic ischemic heart failure in terms of long-term survival.

Bone Marrow or Umbilical Cord Stem Cells Treat Refractory Lupus-Related Kidney Disease

Autoimmune diseases are those diseases in which the patient’s own immune system attacks his or her own tissues. the treatment of such diseases requires giving patients drugs that suppress the immune system. Such drugs have potent side effects and taking such drugs long-term can also predispose patients to cancers and other types of inimical conditions.

One particular type of autoimmune disease, Systemic Lupus Erythematosis, otherwise known as SLE or just Lupus, results from an immune response against components inside our cells. The recognition of these proteins and other substances by our immune system causes massive cell damage and death. However, lupus is a very individual disease. In some patients, the disease manifests by producing butterfly-like lesions on the face.

Lupus butterfly rash (from
Lupus butterfly rash (from

In others, a severe arthritis in several joints results. In other lupus patients, the liver undergoes progressive degradation and scarring. Still others have severe heart problems, and others have scarring and progressive damage to the kidneys. In other patients a combination of symptoms and organs are affected. Some cases of lupus are sporadic and mild, but others are fulminant and relentless. The particular disease a person shows is completely individual.

In some lupus patients, the kidneys experience lupus nephritis (LN), which is inflammation of the tissues of the kidney. In some patients, drug treatments with corticosteroid drugs like prednisone, or other drugs like hydroxychloroquine (Plaquenil), also can help control lupus. Other drugs include powerful immune suppressants such as cyclophosphamide (Cytoxan), azathioprine (Imuran, Azasan), mycophenolate (Cellcept), leflunomide (Arava) and methotrexate (Trexall), all of which have lists of side effects that include increased risk of infection, liver damage, decreased fertility and an increased risk of cancer. However in a percentage of LN patients, drug treatments simply do not work, and these conditions are known as refractory LN.

A new clinical trial has examined the ability of mesenchymal stem cells from bone marrow or umbilical cord tissue to treat refractory LN. This Chinese study examined 81 patients with active refractory LN. The mesenchymal stem cells (MSCs) used in this study were “allogeneic,” which means that they were taken from someone other than the patient. Such treatments have been shown to successfully treat patients with other types of autoimmune diseases (see Figueroa FE, et al., Biol Res. 2012;45(3):269-77).

In this single-center clinical trial, Fei Gu and Dandan Wang and others from the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China enrolled 81 Chinese patients with active and refractory LN from 2007 to 2010. These patients received by intravenous administration either allogeneic bone marrow- or umbilical cord-derived MSCs at a dose of 1 million cells per kilogram of bodyweight. All 81 patients were then monitored over the course of one year with periodic follow-up visits to evaluate kidney function and to determine if the patients were experiencing any adverse events from the stem cell treatments.

During the year-long follow-up, 77 of the 81 patients survived ( survival rate of 95%) and 49/81 patients (60.5%) achieved remission. Eleven of 49 (22.4 %) patients showed a “renal flare,” which means that their symptoms and kidney inflammation returned by the end of 12 months after having previously experienced complete remission.

Kidney function jumped during this time. The main measure of kidney function is a test called the glomerular filtration rate or GFR. This measures how well the kidney filters materials per unit time. GFR in these patients improved significantly 12 months after the stem cell treatment (mean ± SD, from 58.55 ± 19.16 to 69.51 ± 27.93 mL/min). Two other measures used to determine the severity of lupus in a patient (Systemic Lupus Erythematosus Disease Activity Index or SLEDAI score), and the activity of lupus within the kidney (British Isles Lupus Assessment Group or BILAG scores) also decreased consistently, showing that the severity of the disease decreased and the severity of the disease within the kidney also decreased after the stem cell treatment (BILAG scores – 4.48 ± 2.60 at baseline to 1.09 ± 0.83 at 12 month and the SLEDAI scores – 13.11 ± 4.20 at baseline to 5.48 ± 2.77 at 12 months).

If that is not convincing, get this: the doses of prednisone and immunosuppressive drugs required by these patients were tapered. In other words, patients were able to eventually get off their drugs sometime within this year-long period. Is that cool or what!! No transplantation-related adverse events were observed.

Thus, the authors conclude, “Allogeneic MSCT resulted in renal remission for active LN patients within 12-month visit, confirming its use as a potential therapy for refractory LN.”

Now this treatment is NOT a cure. Several patients still experienced renal flares one year after treatment, and not all the patients experienced remission. Therefore, this is not a treatment for everyone. Identifying which patients will be helped by these treatments might require microarray analyses, but the bottom line is clear – some patients are definitely helped by MSC treatments.

Granted this is a small study and it is not a controlled study – these stem cell-treated patients were not compared to anything else. However it is a very hopeful beginning. There were no adverse side effects and 60% of the patients experienced remission, and that is definitely good news

Correcting Mutations Associated with a Blood Disorder

The protein hemoglobin carries oxygen from our lungs to our tissues. Mutations in the genes that encode the protein chains that form hemoglobin can cause inherited blood disorders like sickle-cell anemia, or the so-called Thalassemias. Thalassemias come from the Greek word from sea (θάλασσα or thalassa), because these blood disorders are found in Mediterranean populations. Thalassemias are found in these populations because they convey some resistance to malaria, which was endemic to that area. People with thalassemias tend to have fatigue, weakness, a pale appearance, yellow discoloration of skin (jaundice), facial bone deformities, slow growth, abdominal swelling, or dark urine, although some people have no symptoms.

Now this common genetic blood disorder has been genetically corrected in cultured induced pluripotent stem cells by using cutting-edge genome-editing techniques.

β-Thalassaemia shows reduced levels of hemoglobin, and these reduced levels are due to mutations in the gene that encodes the β-globin protein. Hemoglobin consists of four protein chains, two of which are alpha-globin proteins, and the other two of which are beta-globin proteins. Mutations in the beta-globin gene reduces the levels of functional beta-globin protein and this reduces the levels of functional hemoglobin.

Yuet Kan and his colleagues at the University of California, San Francisco, made induced pluripotent stem cells from skin fibroblasts from a person who suffered from β-thalassemia. Kan and his colleagues then used the CRISPR–Cas9 gene-editing technique to correct the mutation responsible for β-thalassemia. The CRISPR–Cas9 gene-editing technique allows for precise and accurate correction of the mutation without affecting other genes.

After the genetic editing, the iPSCs were differentiated into the precursors of red blood cells in culture and demonstrated that the modified cells showed higher expression of hemoglobin than unmodified cells.

Hopefully transplantation of such corrected cells back into the original patient could one day provide a cure for β-thalassaemia, according to the authors.