Machine pulsatile perfusion for the isolation of Afterbirth-derived stem cells Stem cells are of intense interest for a multitude of therapeutic applications, including tissue repair and for transfusion therapies. For many years, umbilical

cord blood has been used as a non-controversial source of useful stem cells. The use of the entire afterbirth as a source of stem cells can result in a 2-5X improved yield of CD34 positive cells over cord blood alone. Machine pulsatile perfusion is a novel technique for producing afterbirth-derived stem cells which has significant advantages over existing methods, due to the gentle and physiological nature of the pulsatile perfusion process.

Stem cells are characterized by their ability to differentiate into a wide range of specialized cell types. This characteristic of stem cells has energized researchers to focus on the development of stem cell-based therapies, particularly regeneration of tissues, for a wide range of disease targets. Stem cells can be derived from embryonic, fetal, or adult sources. In general, stem cells of embryonic or fetal lineage are considered the most versatile, although there have been recent advances in the transformation of normal adult cells into induced pluripotent stem cells. Despite advances with adult stem cells, most scientists prefer to work with stem cells derived from embryonic or fetal sources because of the flexibility of these cell lines. However, working with these types of tissues is extremely controversial and has been a subject of much political debate over the past decade. For many years, umbilical cord blood (UCB) has been used as a source for hematopoietic stem cells (HSCs). HSCs are stem cells that specifically give rise to blood cell types, including erythrocytes (red blood cells) as well as important components of the immune system, such as lymphocytes, macrophages, platelets, and neutrophils. HSC transplants are now routinely used to treat patients with cancer, as well as other disorders of the blood and immune systems. The use of UCB has significant advantages-- the stem cells derived from cord blood have characteristics of fetal stem cells, and obtaining UCB is non-controversial and non-invasive to the donor. UCB has been a target for allogenic (donor cells from another person) transplants in recent years, since the stem cells in cord blood allow for a less-stringent donor matching procedure as compared to bone marrow blood transplants. Stem cells derived from umbilical cord blood are therefore very useful in helping to reduce the incidence and severity of graft-versus-host disease. Afterbirth-derived stem cells Umbilical cord procedures are limited by low collection volumes of cells. In fact, the yield of stem cells from a single umbilical cord is sufficient for transplants in humans up to only about 70 kg (about 25 lbs). Thus, the banking of umbilical cord blood for later use in the same person (autologous transplant) is inefficient for use in these procedures for anyone other than children under 3 years of age. The placenta has been shown to be a rich source of HSCs as well. In particular, the placenta is a rich source of stem cells that are positive for the marker CD34. CD34+ cell dose is a factor that is has been identified as significantly associated with survival of leukemia patients after stem cell treatment. Hemacell Perfusion, Inc. (HPI) has developed a method to produce afterbirth-derived stem cells (ABDSCs). This method collects stem cells from the entire afterbirth, including placenta and umbilical cord blood. Since the entire afterbirth is used for obtaining stem cells, the yield of cells from this procedure is 2-5X as high as for UCB alone, as demonstrated in Figure 1. “Maximum” and “median” refer to the maximum and median yield of cells from a series of experiments.

Figure 1: Comparison of CD34+ stem cell yield between UCB and ABDSCs obtained by

pulsatile perfusion

Machine Pulsatile Perfusion HPI has developed an innovative method to produce ABDSCs, based on a prototype instrument derived from a kidney perfusion device. This pulsatile method (machine pulsatile perfusion, or MPP), works by extracting the ABDSCs from the entire afterbirth. The method allows for rapid extraction of cells from both placenta and umbilical cord. MPP has a number of advantages over other, non-pulsatile methods: • MPP mimics the action of the human heart, allowing for a smooth transition of blood from the donor

organ to the perfusion circuit. • MPP increases the osmotic pressure of the perfusion solution, thus more efficiently removing placental

blood from the interior of the cells. • The perfusate chemistry can be adjusted to duplicate normal body chemistry, thus extracting placental

blood without effects such as vasoconstriction which may impact stem cell yield. • MPP is more gentle to the placental structure, thus allowing parts of the placenta to be used for human

vascular allografts. All tests performed by HPI to date have demonstrated that ABDSCs isolated by MPP have similar performance to stem cells isolated from UCB with regards to functional white blood cell components and precursors. Future work will include approaches to isolate all useable cells of the placenta, including term amniotic tissue membrane and stem cells derived from the umbilical cord.

In addition to pioneering work in the field of ABDSC, HPI also owns patent rights to an issued patent, US6811965, a kidney perfusion solution containing nitrous oxide which is superior in the preservation of kidneys for transplantation to current best practice. This patent is co-owned by Hemacell and Dr. Youram Vodovotz, who is a practicing transplant surgeon on faculty at the McGowan Institute for Regenerative Medicine in Pittsburgh, PA.

Conclusion

The afterbirth is a rich source of stem cells that can be used in transplantation therapy. Afterbirth-derived stem cells can be obtained in 2-5X the yield of stem cells obtained from umbilical cord blood. Machine pulsatile perfusion, a gentle method that mimics the action of the human heart, is an ideal way to isolate these therapeutically important stem cells.