Tools and Technologies I
Human stem cells hold great potential for use in stem cell-based therapies and regenerative medicine. Technologies developed to date, however, cannot create patient-specific embryonic cell lines, hence transplanting these cells poses innumerable immunological barriers. Another obstacle for embryonic stem cell use is their ability to form tumors. Human placenta is a potential source of primitive fetal stem cells, yet poorly studied. Large volumes of placental tissue could provide abundant numbers of primary stem cells, which will eliminate lengthy ex vivo stem cell propagation. In order to create placental banks, which will allow preservation of each individual’s placental stem cells, technology for the whole placenta cryopreservation ought to be developed. Our hypothesis is that the tissue of human term placenta is a high capacity source of primitive stem cells capable to differentiate into tissues of human body and placenta cryopreservation at birth will provide a life-long supply of own stem cells. In this project, we will demonstrate capability to cryopreserve placentas by means of perfusion with cryopreservation solutions. We will obtain cells from cryopreserved placental tissues and isolate cells expressing surface markers of human stem cells. Cells will be characterized by their morphology, ability to propagate long-term in culture, expression of human embryonic cell-specific markers, engraftment in immunocompromised mice. Use of placental stem cells will greatly reduce the need for embryonic stem cells, which cannot be obtained without destroying human pre-implantation embryos. Placenta as a source of abundant numbers of stem cells will eliminate the need for stem cell line propagation. Cryopreservation of individual placentas would provide life-long source of personal stem cells. As the aims of the application are achieved, creation of the novel placenta stem cell-based therapies will follow.
Statement of Benefit to California:
Stem cell-based therapies are the promising future of medicine. The application of this treatment is limited because of the restricted supply of appropriate stem cell lines, as it is necessary to match certain characteristics of the donor’s and host’s immune systems. Our pioneering studies demonstrated that human placenta may be used as a source of such stem cell lines. These cell lines derived from human placenta will contribute to research in stem cell biology and clinical applications. Furthermore, placentas are readily available from any individual giving birth. In order to create placental banks, which will allow preservation of each individual’s placental stem cells, technology for the whole placenta cryopreservation ought to be developed. Our hypothesis is that the tissue of human term placenta is a high capacity source of primitive stem cells capable to differentiate into tissues of human body, and placenta cryopreservation at birth will provide a life-long supply of own stem cells. As this approach does not require the donation or use of either human embryos or eggs, it will greatly reduce the ethical concerns, and may help overcome the limitations inherent in obtaining excess human embryos. Diseases that can potentially be cured by the use of stem cell treatment afflict significant number of individuals in California. These stem cell lines will be used in regenerative medicine research and cell replacement therapies as well as the development of new treatment approaches. The enhanced and extended lives of the individuals will represent an evident benefit; the savings to the health care system as a consequence of their cure will straightforwardly benefit all California taxpayers. Ultimately the knowledge and experience produced by the work proposed will contribute to the goal of making stem cell transplantation and new medical approaches available to a much broader group of patients, thus greatly extending the benefits to the affected individuals and to the taxpayers of California. Creation of placental banks and cryostorage of human placentas as a source of patient-specific stem cells throughout the life of each individual will some day become a medical necessity, and California may become the pioneer in this breakthrough initiative.
This proposal focuses on the development of methods for cryopreservation of human placentas, to allow harvesting and banking of placental stem cells. The applicants have previously shown that mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) can be derived from human placental tissue. In the first aim they propose to identify an optimal technique for cryopreservation of whole placentas, evaluating both a 4°C system and liquid nitrogen based vitrification. In the second aim they propose to optimize a perfusion system (for preservation) by testing different perfusion solutions, timings and temperatures prior to cryostorage. Finally they propose to confirm the viability and functionality of MSCs and HSCs derived from cyropreserved human placenta, evaluating morphology, marker expression, and differentiation potential. The reviewers did not feel that this proposal addresses a significant roadblock in stem cell biology. They also raised doubts about the project’s feasibility and research design. They recognized the need for banked, autologous pluripotent stem cells but questioned whether placental tissue provides significant advantages over cord blood, a well-established source which is easier to preserve. Better understanding of the basic biology and properties of placental derived cells is needed to more accurately predict the therapeutic utility of these cells and then make more calculated judgment as to whether resources should be allocated to cryopreservation of these cell populations. Additionally, there is practical and technical difficulty of perfusing and freezing placentas on the site at the time of delivery. Time and expertise that will be needed to accomplish that task are not currently available at most birthing centers. Thus there are practical issues that may severely limit the wide-spread implementation of the proposed approach. The reviewers also raised questions about the experimental design. One noted that it lists a number of qualitative criteria for measuring cell viability and functionality following cryopreservation, but it doesn’t include standard measures for assessing cytotoxicity related to cryopreservation. This reviewer also pointed out that in the proposal there is no probe-based monitoring of temperature changes within the placenta during the freezing cycle and there may be marked differences in temperature change rates from placenta to placenta and within a given placenta due to size and shape variations. One reviewer was concerned about the plan to accomplish cryopreserved placentas by placement in a -80°C freezer followed by transfer to liquid nitrogen and wondered why a computer-controlled cryopreservation system was not considered. The reviewer commented,that investigators have underestimated the difficulty of thawing cryopreserved placenta and recovering enough viable cells. Finally, this reviewer also noted that the applicants have underestimated the number of human placenta needed for the study (the power of the study to come up with statistically meaningful results) and felt that many more placentas would be required, due to the large number of variables being studied. The reviewers agreed that the assembled research team is qualified to carry out the work described in the proposal. Some concerns were raised by the over commitment of the PI’s time in other projects. It is not clear that the PI will have sufficient time to complete all projects simultaneously. Overall, the reviewers did not feel that this proposal addresses a significant roadblock to stem cell research. They also raised concerns about feasibility and a potential time over-commitment by the principal investigator.