April 1, 2012 (Vol. 32, No. 7)

Jeffery Herman, Ph.D.

After nearly a decade-long federal moratorium on embryonic stem cell research, regenerative medicine took a serious hit. Yet sometimes from adversity comes innovation. Like-minded regenerative medicine industry leaders illustrated this point at the recent “New York Stem Cell” summit. With novel cell lines involving pure pluripotent embryonic progenitors and “smart” allogenic adult stem cells, to new techniques that include ultrasonic cavitation for the safe, effective separation of the stromal vascular fraction, directional freezing for effective biopreservation of cellular tissue, as well as the clinical application of high-pressurized cultured autologous hyaline cartilage implants, the future of regenerative medicine has never looked brighter.

“There is an old adage in biotech,” said Michael West, Ph.D., CEO of BioTime: “Show me something that I can make.” Since pluripotent embryonic stem cells, by nature, are highly heterogeneous, successfully manufacturing a pure cell product that alleviates all FDA safety concerns has been particularly difficult.

Inspired by Craig Venter, Ph.D.’s approach to sequencing the human genome, BioTime adopted shotgun embryonic cell cloning. By randomly growing a small amount of monoclonal embryonic stem cells with a variety of differentiating signal cues (growth factors such as retinoic acid or bone morphogenetic protein), BioTime developed several embryonic progenitor cells (ACTCellerate™ lines).

ACTCellerate lines exhibit less broad pluripotency but are still relatively undifferentiated with regard to specific downstream tissue. Since ACTCellerate lines are less primitive than embryonic stems cells, they are manufactured with greater purity and still show strong potential for directed clinical therapy against a variety of disease and damaged tissue, Dr. West explained.

Presently BioTime, with the use of microarray, real-time PCR, and protein analysis, has carefully characterized the entire transciptome of over 200 types of ACTCellerate lines in a variety of tissue lineages including chrondogenic, osteogenic, myogenic, and neurogenic cells.

With the process of Fate Space Screening, which involves the random testing of the ACTCellerate lines with different growth factors and culture conditions, BioTime has begun to develop a phylogenetic map of the exact mechanisms and pathways involved in the differentiation of embryonic cells into specific tissues.

While BioTime is interested in the commercialization of its products, it also understands that in order to succeed in the complete and novel elucidation of all tissue differentiation pathways, national and international research collaboration is both a goal and a necessity.

As a way to advance stem cell research and inspire collaboration, BioTime, with Xennex (GeneCards®), is also developing LifeMap, which Dr. West said is the first database to combine known genomic data with the eventual mapping of all development tissue lineage pathways and corresponding signaling mechanisms.


BioTime’s core technologies center on using novel stem cells and hyaluronate-based injectable matrices for applications in regenerative medicine.

Early Lineage Adult Cells

“ELA® cells are genetically and phenotypically distinct from other adult stem cell populations,” noted Pamela Layton, CEO of Parcell Laboratories. They are an early-lineage adult pluripotent cell line found throughout the body, which, according to laboratory studies, can differentiate into endodermal, mesodermal, and ectodermal tissue.

ELA cells lack cell surface biomarkers commonly associated with other stem cell populations and are immune privileged. By not inducing an immune response, ELA cells can be safely donated without the need of HLA matching. Furthermore, with these cells, without the need for immunosuppressant medication, the risk of cellular rejection and graft-versus-host disease may be minimized.

In rats, ELA cells donated from college-aged donors, combined with a demineralized bone scaffold, induced more effective and consistent bone formation than mesenchymal stem cells extracted from college-aged donors, as well as bone marrow aspirate, the “gold standard” in spine repair.

With meticulous donor screening, as well as isolation, processing, and freezing techniques, Layton said that Parcell can successfully harvest large enough quantities of pure ELA cells to not require subsequent cellular expansion. This can result in reduced costs and minimize unnecessary laboratory manipulation.

Layton remarked that Parcell is presently working in collaboration with several top industry leaders to determine the therapeutic applications of ELA cells against many disease indications. In 2010, in collaboration with Alphatec Spine, Parcell developed an ELA-based product, PureGen Osteoprogenitor Cell Allograft™, which has shown “a significant clinical benefit in bone regeneration of the spine.”

Harvesting Stem Cells with Sound

“At the end of the day with our technology, we make more cells, cheaper cells, and faster cells,” said Steven Victor, M.D, CEO of Intellicell Biosciences, when comparing Intellicell technology to other adipose stromal vascular fraction (SVF) harvesting methods.

Intellicell uses ultrasonic cavitation, a phenomenon that occurs with the formation and collapse of microscopic bubbles as a result of an ultrasound pressure gradient. Ultrasonic cavitation strictly utilizes mechanical forces to separate the SVF from white adipose tissue. It does not require the addition of exogenous digestive enzymes, or other purification steps that are common with other SVF harvesting methods.

Additionally, since ultrasonic cavitation lyses red blood cells, the blood does not need to be removed and the “hematopoietic stem cells, plasma, platelets, and other important health factors of the blood” are retained in the SVF population, Dr. Victor said.

Compared to other methods, ultrasonic cavitation provides over twice as many SVF cells from less than half the extracted adipose tissue, with minimal laboratory manipulation. For these reasons, this product falls under FDA 361 guidelines, which will allow the extracted SVF cells to be used off-label and at the discretion of licensed physicians.

Composed of mesenchymal stem cells and other important cell populations, including pre-adipocytes, endothelial cells, smooth muscle cells, fibroblasts, important immune cells, as well as variety of growth factors, the SVF, according to Dr. Victor, is more therapeutically efficacious than stem cells alone. It is pluripotent, exhibits homing characteristics, and can provide clinical regenerative healing in a variety of tissue. Furthermore, since the SVF is extracted from the patient, it is also autologous with minimal risk of an immune response.

Presently Intellicell is largely researching the clinical application of this technology in “three therapeutic worlds”: cosmetics, peridontal medicine, and orthopedics. It is in the process of organizing several clinical trials, including a multicenter study in collaboration with a sports orthopedic medical doctor on the use of ultrasonic cavitation-purified adipose SVF on osteoarthritis of the knee.

To extend its clinical application beyond the focus of Intellicell, the ultrasonic cavitation technology is also licensed worldwide to industry and research leaders to test its clinical effectiveness against other indications including wound healing, cardiac disease, multiple sclerosis, and autism.

Velocity, Key to Tissue Biopreservation

According to Sachi Norman, M.D., CEO of Core Dynamics, the firm’s goal with the use of the Multi-Thermal Gradient (MTG)™ freezing technology is to bring the “science fiction” of cryopreservation into the clinical reality of regenerative medicine.

The precise temperature control needed to properly freeze and store tissue is extremely difficult. It often leads to a significant decrease in cellular viability in response to direct structural damage from random ice crystal formation, as well as indirect damage through changes in cellular signaling, osmotic stress, and chemical toxicity.

MTG takes a different approach to biopreservation with the use of directional freezing. By regulating the velocity of a sample over a steady temperature gradient, well-controlled linear—rather than random—ice crystals can form, which greatly reduces the amount of structural cellular damage in frozen tissue.

The use of toxic chemicals such as DMSO is also not necessary. In preliminary clinical trials, Core Dynamics successfully applied MTG to the clinical cryopreservation and transplantation of osteoarticular grafts into the knee, explained Dr. Norman. It also transplanted fully functional frozen/thawed ovaries, which successfully lead to embryo development in sheep.

With a subsequent sublimation step, Core Dynamics has shown that viable frozen cells can be lyophilized and stored at room temperature. Effective reconstitution of lyophilized cells, due to an upregulation of reactive oxygen species (ROS) and other cellular stress, according to Dr. Norman, is a limiting factor; however, by optimizing the reconstitution fluid with antioxidants and other additives, cord blood stem cells were successfully lyophilized and reconstituted. They exhibited the same biochemical characteristics and increased cell viability compared to normal frozen cord blood cells.

In collaboration with the U.S. Army, Core Dynamics is now working to lyophilize, store, and reconstitute red blood cells. Although not yet clinically viable with a blood cell viability of 70% post-reconstitution, with constant optimization, Core Dynamics is close to perfecting lyophilized blood, said Dr. Norman, which would be a major advantage for healthcare, particularly in regions of the world that cannot afford expensive storage refrigerators and freezers.


A scientist at Core Dynamics demonstrates the operation of one of the company’s directional solidification, multithermal vertical freezing systems, which are based on technology similar to that found in multithermal gradient instruments.

Tissue Engineering and Cell Therapy

Histogenics and ProChon Biotech merged last year, combining tissue engineering with cellular therapy to develop a regenerative medicine company. Kirk Andriano, Ph.D., discussed Histogenics’ NeoCart®, and VeriCart™ technology at the meeting.

NeoCart is an ex vivo implant used to treat large ICRS type III and IV lesions of articular cartilage. The patient’s own cartilage cells are removed and cultured in a high-pressure system that mimics the conditions in the knee while walking.

Once surgically grafted to the damaged cartilage, the implant integrates into the native tissue and induces high-quality hyaline cartilage growth, explained Dr. Andriano. He said that in a recent two-year clinical follow-up study, NeoCart exhibited statistical superiority to microfracture surgery, the presently used gold-standard technique for damaged cartilage.

With microfracture, surgically induced fractures in the subchondral bone plate of the knee create a blood clot, which mediates the growth of weaker fibrous cartilage. NeoCart is presently undergoing Phase III trials and the company is in negotiation with the FDA for a special protocol assessment approval.

VeriCart, on the other hand, according to Dr. Andriano, “will augment microfracture surgery rather than completely replace it.” VeriCart is a cell-free scaffold system rehydrated with autologous bone marrow stem cells and implanted into damaged cartilage.

When combined with microfracture surgery, VeriCart could improve growth and repair of high-quality hyaline cartilage. By using ProChon Biotech’s fibroblast growth factors that influence stem cells to differentiate into early lineage chrondocytes, Histogenics also hopes to develop a one-step procedure that bypasses invasive diagnostic joint surgery, in order to streamline cartilage repair.


NeoCart training sample in a petri dish. NeoCart is an ex vivo implant used to treat large ICRS type III or IV lesions of articular cartilage. [Histogenics]

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