Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.
Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.
Until recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic “somatic” or “adult” stem cells. The functions and characteristics of these cells will be explained in this document. Scientists discovered ways to derive embryonic stem cells from early mouse embryos more than 30 years ago, in 1981. The detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures. When they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be “reprogrammed” genetically to assume a stem cell-like state. This new type of stem cell, called induced pluripotent stem cells (iPSCs), will be discussed in a later section of this document.
Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, the inner cells give rise to the entire body of the organism, including all of the many specialized cell types and organs such as the heart, lungs, skin, sperm, eggs and other tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury, or disease.
- Hematopoietic stem cells[rx] give rise to all the types of blood cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.
- Mesenchymal stem cells[rx] have been reported to be present in many tissues. Those from bone marrow (bone marrow stromal stem cells, skeletal stem cells) give rise to a variety of cell types: bone cells (osteoblasts and osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and stromal cells that support blood formation. However, it is not yet clear how similar or dissimilar mesenchymal cells derived from non-bone marrow sources are to those from bone marrow stroma.
- Neural stem cells[rx] in the brain give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal cells—astrocytes and oligodendrocytes.
- Epithelial stem cells – in the lining of the digestive tract occur in deep crypts and give rise to several cell types: absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells.
- Skin stem cells – occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis.
A. What stages of early embryonic development are important for generating embryonic stem cells?
Embryonic stem cells, as their name suggests, are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro—in an in vitro fertilization clinic—and then donated for research purposes with the informed consent of the donors. They are not derived from eggs fertilized in a woman’s body.
B. How are embryonic stem cells grown in the laboratory?
Growing cells in the laboratory are known as cell culture. Human embryonic stem cells (hESCs) are generated by transferring cells from a preimplantation-stage embryo into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. In the original protocol, the inner surface of the culture dish was coated with mouse embryonic skin cells specially treated so they will not divide. This coating layer of cells is called a feeder layer. The mouse cells at the bottom of the culture dish provide the cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Researchers have now devised ways to grow embryonic stem cells without mouse feeder cells. This is a significant scientific advance because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
The process of generating an embryonic stem cell line is somewhat inefficient, so lines are not produced each time cells from the preimplantation-stage embryo are placed into a culture dish. However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed gently and plated into several fresh culture dishes. The process of re-plating or subculturing the cells is repeated many times and for many months. Each cycle of subculturing the cells is referred to as a passage. Once the cell line is established, the original cells yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line. At any stage in the process, batches of cells can be frozen and shipped to other laboratories for further culture and experimentation.
C. What laboratory tests are used to identify embryonic stem cells?
At various points during the process of generating embryonic stem cell lines, scientists test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells. This process is called characterization.
Scientists who study human embryonic stem cells have not yet agreed on a standard battery of tests that measure the cells’ fundamental properties. However, laboratories that grow human embryonic stem cell lines use several kinds of tests, including:
- Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term growth and self-renewal. Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated.
- Using specific techniques to determine the presence of transcription factors that are typically produced by undifferentiated cells. Two of the most important transcription factors are Nanog and Oct4. Transcription factors help turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. In this case, both Oct 4 and Nanog are associated with maintaining the stem cells in an undifferentiated state, capable of self-renewal.
- Using specific techniques to determine the presence of particular cell surface markers that are typically produced by undifferentiated cells.
- Examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed. It does not detect genetic mutations in the cells.
- Determining whether the cells can be re-grown, or subcultured, after freezing, thawing, and re-plating.
- Testing whether the human embryonic stem cells are pluripotent by 1) allowing the cells to differentiate spontaneously in cell culture; 2) manipulating the cells so they will differentiate to form cells characteristic of the three germ layers; or 3) injecting the cells into a mouse with a suppressed immune system to test for the formation of a benign tumor called a teratoma. Since the mouse’s immune system is suppressed, the injected human stem cells are not rejected by the mouse immune system and scientists can observe growth and differentiation of the human stem cells. Teratomas typically contain a mixture of many differentiated or partly differentiated cell types—an indication that the embryonic stem cells are capable of differentiating into multiple cell types.
National Institutes of Health Guidelines for Research Using Human Stem Cells
Scope of Guidelines
These Guidelines apply to the expenditure of National Institutes of Health (NIH) funds for research using human embryonic stem cells (hESCs) and certain uses of induced pluripotent stem cells (See Section IV). The Guidelines implement Executive Order 13505.
Long-standing HHS regulations for Protection of Human Subjects, 45 C.F.R. 46, Subpart A establish safeguards for individuals who are the sources of many human tissues used in research, including non-embryonic human adult stem cells and human-induced pluripotent stem cells. When research involving human adult stem cells or induced pluripotent stem cells constitutes human subject research, Institutional Review Board review may be required and informed consent may need to be obtained per the requirements detailed in 45 C.F.R. 46, Subpart A. Applicants should consult http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html.
It is also important to note that the HHS regulation, Protection of Human Subjects, 45 C.F.R. Part 46, Subpart A, may apply to certain research using hESCs. This regulation applies, among other things, to research involving individually identifiable private information about a living individual, 45 C.F.R. § 46.102(f). The HHS Office for Human Research Protections (OHRP) considers biological material, such as cells derived from human embryos, to be individually identifiable when they can be linked to specific living individuals by the investigators either directly or indirectly through coding systems. Thus, in certain circumstances, IRB review may be required, in addition to compliance with these Guidelines. Applicant institutions are urged to consult OHRP guidance at http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html
To ensure that the greatest number of responsibly derived hESCs are eligible for research using NIH funding, these Guidelines are divided into several sections, which apply specifically to embryos donated in the U.S. and foreign countries, both before and on or after the effective date of these Guidelines. Section II (A) and (B) describe the conditions and review processes for determining hESC eligibility for NIH funds. Further information on these review processes may be found at www.NIH.gov. Sections IV and V describe research that is not eligible for NIH funding.
These guidelines are based on the following principles:
- Responsible research with hESCs has the potential to improve our understanding of human health and illness and discover new ways to prevent and/or treat illness.
- Individuals donating embryos for research purposes should do so freely, with voluntary and informed consent.
As directed by Executive Order 13505, the NIH shall review and update these Guidelines periodically, as appropriate.
Eligibility of Human Embryonic Stem Cells for Research with NIH Funding
For the purpose of these Guidelines, “human embryonic stem cells (hESCs)” are cells that are derived from the inner cell mass of blastocyst-stage human embryos, are capable of dividing without differentiating for a prolonged period in culture, and are known to develop into cells and tissues of the three primary germ layers. Although hESCs are derived from embryos, such stem cells are not themselves human embryos. All of the processes and procedures for review of the eligibility of hESCs will be centralized at the NIH as follows:
Applicant institutions proposing research using hESCs derived from embryos donated in the U.S. on or after the effective date of these Guidelines may use hESCs that are posted on the new NIH Registry or they may establish eligibility for NIH funding by submitting an assurance of compliance with Section II (A) of the Guidelines, along with supporting information demonstrating compliance for administrative review by the NIH. For the purposes of this Section II (A), hESCs should have been derived from human embryos:
- That were created using in vitro fertilization for reproductive purposes and was no longer needed for this purpose; that were donated by individuals who sought reproductive treatment (hereafter referred to as “donor(s)”) and who gave voluntary written consent for the human embryos to be used for research purposes; and for which all of the following can be assured and documentation provided, such as consent forms, written policies, or other documentation, provided:
- All options available in the health care facility where treatment was sought pertaining to the embryos no longer needed for reproductive purposes were explained to the individual(s) who sought reproductive treatment.
- No payments, cash or in-kind, were offered for the donated embryos.
- Policies and/or procedures were in place at the health care facility where the embryos were donated that neither consenting nor refusing to donate embryos for research would affect the quality of care provided to a potential donor(s).
There was a clear separation between the prospective donor(s)’s decision to create human embryos for reproductive purposes and the prospective donor(s)’s decision to donate human embryos for research purposes. Specifically:
- Decisions related to the creation of human embryos for reproductive purposes should have been made free from the influence of researchers proposing to derive or utilize hESCs in research. The attending physician responsible for reproductive clinical care and the researcher deriving and/or proposing to utilize hESCs should not have been the same person unless separation was not practicable.
- At the time of donation, consent for that donation should have been obtained from the individual(s) who had sought reproductive treatment. That is, even if the potential donor(s) had given a prior indication of their intent to donate to research any embryos that remained after reproductive treatment, consent for the donation for research purposes should have been given at the time of the donation.
- Donor(s) should have been informed that they retained the right to withdraw consent for the donation of the embryo until the embryos were actually used to derive embryonic stem cells or until information which could link the identity of the donor(s) with the embryo was no longer retained, if applicable.
During the consent process, the donor(s) were informed of the following:
- that the embryos would be used to derive hESCs for research;
- what would happen to the embryos in the derivation of hESCs for research;
- that hESCs derived from the embryos might be kept for many years;
- that the donation was made without any restriction or direction regarding the individual(s) who may receive medical benefit from the use of the hESCs, such as who may be the recipients of cell transplants.;
- that the research was not intended to provide direct medical benefit to the donor(s);
- that the results of research using the hESCs may have commercial potential, and that the donor(s) would not receive financial or any other benefits from any such commercial development;
- whether information that could identify the donor(s) would be available to researchers.
Applicant institutions proposing research using hESCs derived from embryos donated in the U.S. before the effective date of these Guidelines may use hESCs that are posted on the new NIH Registry or they may establish eligibility for NIH funding in one of two ways:
- By complying with Section II (A) of the Guidelines; or
- By submitting materials to a Working Group of the Advisory Committee to the Director (ACD), which will make recommendations regarding eligibility for NIH funding to its parent group, the ACD. The ACD will make recommendations to the NIH Director, who will make final decisions about eligibility for NIH funding.
- The materials submitted must demonstrate that the hESCs were derived from human embryos: 1) that were created using in vitro fertilization for reproductive purposes and were no longer needed for this purpose; and 2) that were donated by donor(s) who gave voluntary written consent for the human embryos to be used for research purposes.
- The Working Group will review submitted materials, e.g., consent forms, written policies or other documentation, taking into account the principles articulated in Section II (A), 45 C.F.R. Part 46, Subpart A, and the following additional points to consider. That is, during the informed consent process, including written or oral communications, whether the donor(s) were: (1) informed of other available options pertaining to the use of the embryos; (2) offered any inducements for the donation of the embryos; and (3) informed about what would happen to the embryos after the donation for research.
- For embryos donated outside the United States before the effective date of these Guidelines, applicants may comply with either Section II (A) or (B). For embryos donated outside of the United States on or after the effective date of the Guidelines, applicants seeking to determine eligibility for NIH research funding may submit an assurance that the hESCs fully comply with Section II (A) or submit an assurance along with supporting information, that the alternative procedural standards of the foreign country where the embryo was donated provide protection at least equivalent to those provided by Section II (A) of these Guidelines. These materials will be reviewed by the NIH ACD Working Group, which will recommend to the ACD whether such equivalence exists. Final decisions will be made by the NIH Director.
- NIH will establish a new Registry listing hESCs eligible for use in NIH funded research. All hESCs that have been reviewed and deemed eligible by the NIH in accordance with these Guidelines will be posted on the new NIH Registry.
Use of NIH Funds
Prior to the use of NIH funds, funding recipients should provide assurances, when endorsing applications and progress reports submitted to NIH for projects using hESCs, that the hESCs are listed on the NIH registry.
Research Using hESCs and/or Human Induced Pluripotent Stem Cells That, Although the Cells May Come from Eligible Sources, is Nevertheless Ineligible for NIH Funding
This section governs research using hESCs and human-induced pluripotent stem cells, i.e., human cells that are capable of dividing without differentiating for a prolonged period in culture and are known to develop into cells and tissues of the three primary germ layers. Although the cells may come from eligible sources, the following uses of these cells are nevertheless ineligible for NIH funding, as follows:
- Research in which hESCs (even if derived from embryos donated in accordance with these Guidelines) or human induced pluripotent stem cells are introduced into non-human primate blastocysts.
- Research involving the breeding of animals where the introduction of hESCs (even if derived from embryos donated in accordance with these Guidelines) or human induced pluripotent stem cells may contribute to the germline.
Other Research Not Eligible for NIH Funding
- NIH funding of the derivation of stem cells from human embryos is prohibited by the annual appropriations ban on funding of human embryo research (Section 509, Omnibus Appropriations Act, 2009, Pub. L. 111-8, 3/11/09), otherwise known as the Dickey Amendment.
- Research using hESCs derived from other sources, including somatic cell nuclear transfer, parthenogenesis, and/or IVF embryos created for research purposes, is not eligible for NIH funding.