The Wild Card of Pluripotency - The Egg

The causality dilemma of what comes first has perplexed our conscious brains and imbued scope to our circular conversations of how. This notion of preconceived programming, intelligent yet evolving, within a time continuum of universal dimensions always brings us back to the intersection of life's origins and its elements. What is consistent in this saga of reality and human perception is that there are new beginnings where The Egg plays a central role - a protagonist of life, without which, for the moment, we wouldn't be.

This thought provoking origin story, fascinating as it is, distracts from the practical issues of the here and now where scientific investigators are just beginning to fathom the power of a woman's fertility process - be it amniotic fluid, menstrual blood, neonatal membrane, umbilical cords or The Egg itself and all the wondrous micro molecules it holds.

Regenerative medicine and those that search for the answers to our ills have long sought the primordial knowledge within the woman's body to the process of reproduction. That sacred temple from which life emerges in all its form and function. The "V" mainframe of life's blueprint in regenerative science. Call it the black box if you like but it holds the holy grail of developmental knowledge for man - the how in practical terms.

While controversial to some the use of Eggs to research and develop cellular solutions is necessary as a player in the age old two step union and potentially as an invaluable starting source for embryonic cells via a non-fertilization development pathway. This process is called Parthenogenesis and is the unique domain of International Stem Cell Corporation ("ISCO").



ISCO was formed as a continuation of scientific endeavors establishing primary cell technology and regenerative therapies for the skincare market. As early leaders in the research business they established the foundation and management reached out to pioneering Russian scientists to employ proprietary techniques in the use of Eggs for parthenogenetically derived pluripotent stem cell lines for therapeutics. The company's focus on chemically activating Eggs to stimulate cell division without fertilizing side-stepped the then burning issue of the use of embryos in science. Independence and solid support from the scientific management team has brought the technology front and center with the start of the first parthenogenetic derived stem cell trial for Parkinson's disease.

Having followed ISCO for some time I reached out the Dr. Russell Kern, ISCO's CSO, for a Q&A on their technology, the data and progress the company has made getting it's lead program into the clinic and to review where things stand overall as they move ahead with proof of concept.

Cheers        

Q&A       

1. The science of parthenogenetic pluripotency, your company’s core focus, is closely linked with SCNT and the early work on developing reprogramming technology using activated human eggs without fertilizing them. To differentiate the two technologies, parthenogenetics versus SCNT, would you briefly review for us the unique science techniques involved here?

In somatic cell nuclear transfer (SCNT), the nucleus of an oocyte is replaced by the nucleus of a somatic cell¹. Once the somatic nucleus is inserted into the oocyte, the nucleus is reprogrammed by the host egg cell and is stimulated to divide to reach a blastocyst stage. This activated egg is viable and capable of producing an adult organism with all the necessary genetic information. Human embryonic stem cells are isolated from the inner cell mass of the blastocyst for use in cell therapy or disease modeling.

Parthenogenesis consists of the chemical activation of an unfertilized oocyte. The activated oocyte starts to divide to reach a blastocyst like state and eventually becomes diploid but it is not capable of producing an adult organism. Human parthenogenetic stem cells are isolated from the inner cell mass of this blastocystlike state, they are also pluripotent and can be usedfor cell therapy and drug screening.

2. With regard to your therapeutic programs, could you outline the clinical and preclinical work you are engaged in and the rough pipeline schedule you envision moving forward?

ISCO’s Phase I study evaluating the safety and tolerability of ISChpNSCs for Parkinson’s disease is currently being conducted in Australia and we plan to move forward with a Phase II study in Australia and USA.

The company also has a traumatic brain injury (TBI) program in development which uses the same ISChpNSC cells. The program is under preclinical development and we hope to complete it and file for regulatory approval in the near future.

ISCO is also working in osteoarthritis. The osteoarthritis program is an autologous cell therapy. We will use the patient’s own adipose tissue to derive chondrocyte for cartilage repair. We will use our 3D bioprinting technology to print cartilage tissue tailored to patient’s specific needs.

3. The Parkinson’s Disease program is now enrolling patients in Australia for a first in man trial using pluripotent stem cell. This achievement has excited the patient community and raised questions from your neuroscientist colleagues in the field. Would you kindly address the pertinent issues related to those published comments² and speak to the patient community with regard to the proposed trials in development.

a. What is being transplanted, and what is the proposed mechanism of action?

The clinical trial will be transplanting human parthenogenetic stem cells (ISChpNSCs). Human parthenogenetic stem cells are pluripotent stem cells that express all the pluripotent stem cell markers and, more importantly, have the ability to differentiate into all three germ layers, including dopaminergic neurons. The therapeutic potential of parthenogenetic stem cells derived cells have been tested by ISCO and other laboratories in various animal disease models, including Parkinson’s disease (PD).

ISCO’s preclinical work indicates that the proposed mechanism of action of ISChpNSCs is neurotrophic support and cell replacement to the dying dopaminergic neurons of the recipient PD brain. The ISChpNSCs secrete neurotrophic cytokines in vitro and have been shown to significantly increase the levels of these cytokines in vivo³.

ISCO has shown that ISChpNSCs differentiate into dopaminergic (DA) neurons (TH+, FOXA2+, GIRK2+, and DAT+) that secrete dopamine and fire spontaneous action potentials³.

Additionally, in a recent publication from Roger Barker’s lab titled “PAX6 expression may be protective against dopaminergic cell loss in Parkinson’s disease” they found that the transcription factor, PAX6 is expressed in midbrain dopaminergic neurons and concluded that it may be protective against dopaminergic cell loss in Parkinson’s disease.

We have shown that ISChpNSCs also differentiate in situ into DA neurons in two different PD animal models, 6O HDA lesioned rats and MPTP lesioned African green monkeys.  The percentage of ISChpNSCs differentiating into DA neurons in vivo is around 1 to 2% of the engrafted cells, which is enough to have a significant increase in the total number of DA neurons in the substantia nigra and fiber innervation in the striatum.

b. What are the preclinical safety and efficacy data supporting the use of the proposed stem cell product?

ISCO has published and presented its preclinical data at multiple international scientific conferences including International Society for Stem Cell Research, American Academy of Neurology, American Neurological Association, Society for Neuroscience, International Society for Cell Therapy, International Society for Neural Therapy and Repair, and the American Society of Gene and Cell Therapy.

In addition to the 10 rats and the 2 monkey study mentioned in the commentary, we have completed a long term 12 month transplantation study of hpNSCs in 20 MPTP lesioned African green monkeys with moderate to severe clinical Parkinsonian symptoms. The ISChpNSCs were manufactured under current good manufacturing practice (cGMP) conditions and were injected bilaterally into the striatum and substantia nigra of immunosuppressed monkeys. Transplantation of ISChpNSC was found to be safe and well tolerated by the animals. We observed higher behavioral recovery, dopamine levels, innervation and number of dopaminergic neurons than in vehicle control animals.

c. Can arguments concerning ethics, risk mitigation, or trial logistics outweigh concerns regarding the expected efficacy of the cell and constitute a primary justification for choosing one cell type over another in a clinical trial?

The assumption that the study received approval based on ethical considerations is incorrect.

The ISChpNSC line has undergone stringent quality control measures and has received FDA approval to be used for IND submission in clinical trials. 

The Australian Therapeutics Goods Administration (TGA), which is FDA’s Australian counterpart and the Human Research Ethics Committee (HREC) have reviewed ISCO’s extensive preclinical data and decided to grant approval for its treatment of Parkinson’s disease. The ISChpNSCs are a more practical source of cells than obtaining fetal cells from aborted human fetal brains. ISCO can manufacture an unlimited supply of homogenous populations of ISChpNSCs under cGMP conditions and preclinical studies indicate that they are safe and promote behavioral and biochemical functional improvements in a nonhuman primate Parkinson’s disease model.

d. What is being claimed regarding the potential therapeutic value of the stem cellbased therapy – better control of symptoms or a cure?

ISCO claims that preclinical studies have shown improvement in PD symptoms and increase in brain dopamine levels following the intracranial administration of ISChpNSC.

e. What is the regulatory oversight of the trial and is it guided by input from experts in the field?

The Australian regulatory agency, TGA, is in possession of the criteria and expertise for evaluating preclinical studies and clinical investigators and members of ethics committees are properly disposed to evaluate complex scientific data and thereby weigh risk against potential benefit on behalf of PD patients.

It is worth noting that ISCO’s trial is fully funded by the company with no economic involvement from patients. ISCO’s trial has a five year follow up with a number of safety and efficacy measures, including Fdopa positron emission tomography (PET) scans.

4. In regard to the cell type being used in your PD trial are there any specific inherent advantages or disadvantages in using parthenogenetic derived cells as opposed to other cell types for Parkinson’s Disease?

The derivation of human parthenogenetic stem cells (hpSCs) does not involve the destruction of a human embryo, so they are free of the ethical concerns associated with hESCs. They can be produced in abundant quantities which will be important in treating patients. The hpSCs can be derived homozygous at the HLA loci from both heterozygous and homozygous donors. HLA homozygous lines have the potential to immune match millions of patients in cell based therapy applications if the HLA type is common. For that reason, they are a promising choice for regenerative medicine applications.

Additionally, a recent study comparing the frequencies of coding mutations in human pluripotent stem cells has also found that hpSCs have lower number of de novo coding mutations than iPSCs and NTESCs.

5. In a recent news release there was mention of additional cell types in development, one of which was neurons specifically for the Parkinson’s program, can you elaborate on that?

ISCO is using human parthenogenetic derived neural stem cells (ISChpNSC) for its Parkinson’s and Stroke programs but not neurons.

6. Why is ISCO not part of the consortium called GForce PD, joining the collaborative effort to share development data on Parkinson’s therapeutic research?

GForce PD is formed by nonprofit academic institutions. ISCO, being a for profit biotechnology company, cannot be as open to share research and development data for intellectual property reasons. We try to the best of our ability to share our data with the appropriate forums to the extent that we can without compromising our work. For example, we frequently present preclinical data at multiple international scientific conferences including International Society for Stem Cell Research, American Academy of Neurology, American Neurological Association, Society for Neuroscience, International Society for Cell Therapy, International Society for Neural Therapy and Repair, and the American Society of Gene and Cell Therapy among others.

7. Being a California company have you applied for CIRM support funding for your programs? If so was there a reason CIRM did not approve support funding given the potential? 

We were not able to apply for CIRM funding for our clinical trial in the Australia because CIRM funding can only be used to conduct clinical trials in California.

8. Is there an update you can share on the enrollment in Australia for the PD trial? Have you completed enrollment as anticipated? Has Foley started treatment procedures and when do they expect to complete the cohorts? 

Enrollment has started and dosing is anticipated to be completed in 2016.

9. You have indicated that the FDA has approved your parthenogenetic cell line for clinical use in the USA. Can you provide an overview of those discussions and the approval process in light of the questions regarding cell line safety data and US trial plans? 

The ISChpNSC line has undergone extensive testing and stringent quality control measures and has received permission from the FDA for use in clinical trials under the Investigative New Drug (IND) application procedure. Critical to the approval for clinical use was the data package provided to the FDA in which ISCO summarized the methods, standards and results of the testing performed on the cell line. The data package was developed as a result of the company’s discussions with the FDA to clarify the safety and acceptability of the proposed cell line for therapeutic use in clinical trials for Parkinson’s disease.

10. You’ve highlighted the creation of a parthenogenetic stem cell bank as one of the keys to unlocking the potential to better match patients to your derived treatments, as is the case also with other pluripotent stem cell sources. Can you review the topic and progress made to establish the UniStemCell bank?

UniStemCell bank is the life science industry’s first collection of histocompatible parthenogenetic stem cells available for research and commercial use. ISCO uses its proprietary technique of parthenogenesis to create histocompatible stem cells. These cells have a duplicate set of human leukocyte antigen (HLA) genes which significantly reduces the possibility that they will be rejected by an individual’s immune system, making a single cell line suitable for treating millions of individuals. An additional benefit is that a relatively small number of such cell lines could be sufficient to provide “immune matched” cells to a large percentage of the world’s population.

When used for cell based therapies, stem cells face the same HLA matching issues that limit solid organ allogeneic transplants and lead to immune rejection. The risk of rejection is proportional to the degree of disparity between donor and recipient cell surface antigen presenting proteins. ISCO is continuing the development of the UniStemCell bank.

11. Recently haploid stem cells were isolated using parthenogenetic techniques. Can you comment on this development and its impact on your science and intellectual property position?

In the recent paper published in Nature, Sagi and colleagues isolated and maintained for the first time human pluripotent stem cell lines with a normal haploid karyotype. They found that some human parthenogenetic stem cell lines have a small percentage of haploid cells in culture and they were able to isolate these cells and expand them in culture to obtain a population of haploid pluripotent stem cells. These haploid stem cells are useful for studying human functional genomics and development but they are unlikely to be used for cell therapy applications because they spontaneously become diploid, at a rate of 3–9% cells per day, and require constant sorting to maintain a population of haploid cells.

12. Human eggs are required for the creation of parthenogenetic stem cell lines which in some circles raised a red flag on the ethics of use and the safety of women undergoing the now standard IVF fertility treatment stimulation procedure. Can you address this issue, the scope of donations required to establish a working stem cell bank and the need for egg donors moving forward given possible reprogramming technology advances.

Donation of oocytes to ISCO occurs under informed consent (IC), and with the approval and oversight of an institutional review board (IRB), both of which provide assurance that the donor is fully aware of the IVF procedures, risks, purpose, potential benefits and voluntary nature of the donation. Since the donation involves eggs left over from IVF, the donor is at no higher risk for participating than she would otherwise experience from the procedure itself.

A single parthenogenetic stem cell line can be expanded to generate large numbers of cells in the form of Master and Working Cell Banks (MCB and WCB) which are cryopreserved to maintain a viable repository over an extended period of time. The ability to amplify and preserve large populations of clonally derived cells in this manner is advantageous for maintaining consistency of the clinical product, as well as for minimizing the need for a continuous source of donated oocytes. Oocytes do not fall into the controversial category of embryos and hence the ethical concerns associated with the use of embryonic stem cells do not apply to parthenogenetic stem cells.

13. Lastly I’d like to address the issue of the uplist from the OTC and finances. In your recent prospectus it was stated that you will look to uplist to Nasdaq – is that still the case and if so when will that occur? Also, of late you have been largely self funded through Dr. Andrey Semechkin, CEO and Co-Chairman of the company. Could you speak to the issue of choosing to back the company with insider funding support, business development plans for program partnerships and the longer term strategy given the requirements of your core business to bring therapeutic stem cell treatments to market?

Our focus right now is to do great work with our clinical trial in Australia as well as advance our other development programs in the pipeline. We believe that the way the company is structured now makes a lot of good business sense and we don’t have any plans to change things any time soon. Our management is fully committed to see our different programs to fruition and is fully confident in our technology and therefore decided to further invest in the company.


We believe we are well funded to get a few of our programs to the next milestone. At different times of development, the company has had discussions with potential partners and we may very well decide to partner with another company as we prepare to bring products to market. However, currently we are focused on moving our development programs forward.

References:

1. Tachibana, M. , et al. Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 153, 12281238 (2013).

2. Barker, R.A. , et al. Are Stem CellBased Therapies for Parkinson's Disease Ready for the Clinic in 2016? Journal of Parkinson's disease 6, 5763 (2016).

3. Gonzalez, R. , et al. Deriving dopaminergic neurons for clinical use. A practical approach. Scientific Reports 3, 15 (2013).

4. Thomas, M.G. , et al. PAX6 expression may be protective against dopaminergic cell loss in Parkinson's disease. CNS & neurological disorders drug targets 15, 7379 (2016).

5. Gonzalez, R. , et al. Proof of concept studies exploring the safety and functional activity of human parthenogeneticderived neural stem cells for the treatment of Parkinson's disease. Cell transplantation 24, 681690 (2015).

6. Johannesson, B. , et al. Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors. Cell stem cell 15, 634642 (2014).

7. Sagi, I. , et al. Derivation and differentiation of haploid human embryonic stem cells. Nature (2016).