Stem cell biology is a rapidly expanding field that explores the characteristics and possible clinical applications of a variety of stem cells that serve as the progenitors of more differentiated cell types. In addition to potential therapeutic applications (Chap. 90e), patient-derived stem cells can also be used as disease models and as a means of testing drug efficacy. Stem cells and their niche are a major focus of medical research because they play central roles in tissue and organ homeostasis and repair, which are important aspects of aging and disease.
IDENTIFICATION, ISOLATION, AND DERIVATION OF STEM CELLS
The definition of stem cells remains elusive. Stem cells were originally postulated as unspecified or undifferentiated cells that provide a source of renewal of skin, intestine, and blood cells throughout life. These resident stem cells have been identified in a variety of organs (e.g., epithelia of the skin and digestive system, bone marrow, blood vessels, brain, skeletal muscle, liver, testis, and pancreas) based on their specific locations, morphology, and biochemical markers.
Unequivocal identification of stem cells requires their separation and purification, usually based on a combination of specific cell-surface markers. These isolated stem cells (e.g., hematopoietic stem [HS] cells) can be studied in detail and used in clinical applications, such as bone marrow transplantation (Chap. 89e). However, the lack of specific cell-surface markers for other types of stem cells has made it difficult to isolate them in large quantities. This challenge has been partially addressed in animal models by genetically marking different cell types with green-fluorescent protein driven by cell-specific promoters. Alternatively, putative stem cells have been isolated from a variety of tissues as side population (SP) cells using fluorescence-activated cell sorting after staining with the Hoechst 33342 dye.
It is desirable to culture and expand stem cells in vitro to obtain a sufficient quantity for analysis and potential therapeutic use. Although the derivation of stem cells in vitro has been a major obstacle in stem cell biology, the number and types of cultured stem cells have increased progressively (Table 88-1). Cultured stem cells derived from resident stem cells are often called adult stem cells or somatic stem cells to distinguish them from embryonic stem (ES) and embryonic germ (EG) cells. However, considering the existence of embryo-derived, tissue-specific stem cells (e.g., trophoblast stem [TS] cells) and the possible derivation of similar cells from an embryo/fetus (e.g., neural stem [NS] cells), it is more appropriate to use the term, tissue stem cells.
TABLE 88-1Examples of Cultured Stem Cells ||Download (.pdf) TABLE 88-1Examples of Cultured Stem Cells
|Name ||Source |
|Embryonic stem cells (ES, ESC) ||Blastocysts or immunosurgically isolated inner cell mass (ICM) from blastocysts |
|Embryonic germ cells (EG, EGC) ||Primordial germ cells (PGCs) from embryos at E8.5–E12.5 (m); gonadal tissues from 5–11 week postfertilization embryo/fetus (h) |
|Trophoblast stem cells (TS, TSC) ||Trophectoderm of E3.5 blastocysts, extraembryonic ectoderm of E6.5 embryos, and chorionic ectoderm of E7.5 embryos (m) |
|Extraembryonic endoderm cells (XEN) ||ICM from blastocysts (m) |
|Embryonal carcinoma cells (EC) ||Teratocarcinoma—a type of cancer that develops in the testes and ovaries (m, h) |
|Mesenchymal stem cells (MS, MSC) ||Bone marrow, muscle, adipose tissue, peripheral blood, and umbilical cord blood (m, h) |
|Multipotent adult stem cells (MAPC) ||Bone marrow mononuclear cells (m, h); postnatal muscle and brain (m) |
|Spermatogonial stem cells (SS, SSC) ||Newborn testis (m) |
|Germline stem cells (GS, GSC) ||Neonatal testis (m) |
|Multipotent adult germline stem cells (maGSC) ||Adult testis (m) |
|Neural stem cells (NS, NSC) ||Fetal and adult brain (subventricular zone, ventricular zone, and hippocampus) (m, h) |
|Unrestricted somatic stem cells (USSC) ||Mononuclear fraction of cord blood (h) |
|Epistem cells (EpiSC) ||Early postimplantation epiblast (m) |
|Induced pluripotent stem cells (iPS, iPSC) ||Variety of terminally differentiated cells and tissue stem cells (m, h) |
|Multilineage-differentiating stress-enduring (MUSE) cells ||Adult human bone marrow stromal cells and dermal fibroblasts |
|Urine-derived stem cells (USC) ||Urine (h) |
|Lung stem cells ||Lung (m, h) |
|Amniotic fluid-derived stem (AFS) cells ||Amniotic fluid (m, h) |
|Umbilical cord blood stem cells ||Umbilical cord (h) |
|Adipose stem cells (AST) ||Fat (m, h) |
|Cardiac stem cells ||Heart (m, h) |
|Renal stem cells ||Renal papilla (m, h) |
|Crypt stem cells ||Intestine (m, h) |
|Colon stem cells (CoSC) ||Colon (m, h) |
|Hepatic stem cells ||Liver (m, h) |
|Dental pulp stem cells (DPSC) ||Dental pulp (m, h) |
|Hair follicle stem cells ||Hair (m, h) |
Successful derivation of cultured stem cells (both embryonic and tissue stem cells) often requires the identification of necessary growth factors and culture conditions, mimicking the microenvironment or niche of the resident stem cells. Recently, long-term maintenance of tissue stem cells in vitro is increasingly possible by growing them as three-dimensional (3D) organoids, which contain both stem cells and niche cells (Chap. 92e). For example, intestinal stem cells can now be cultured as “epithelial mini-guts” in the presence of R-spondin, epidermal growth factor (EGF), and noggin on Matrigel. Similarly, lung stem cells can be cultured as self-renewing “alveolospheres.” A growing list of cultured stem cells, although not comprehensive, is shown in Table 88-1. Please note that the establishment of cultured stem cells is often under dispute due to the difficulties in assessing the characteristics of these cells.
SELF-RENEWAL AND PROLIFERATION OF STEM CELLS
Symmetric and Asymmetric Cell Division
The most widely accepted stem cell definition is a cell with a unique capacity to produce unaltered daughter cells (self-renewal) and to generate specialized cell types (potency). Self-renewal can be achieved in two ways. Asymmetric cell division produces one daughter cell that is identical to the parental cell and one daughter cell that is different from the parental cell and is a progenitor or differentiated cell. Asymmetric cell division does not increase the number of stem cells. Symmetric cell division produces two identical daughter cells. For stem cells to proliferate in vitro, they must divide symmetrically.
Unlimited Expansion In Vitro
Resident stem cells are often quiescent and divide infrequently. However, once the stem cells are successfully cultured in vitro, they often acquire the capacity to divide continuously and the ability to proliferate beyond the normal passage limit typical of primary cultured cells (sometimes called immortality). These features are primarily seen in ES cells but have also been demonstrated for tissue stem cells, such as NS cells and mesenchymal stem (MS) cells, thereby enhancing the potential of these cells for therapeutic use (Table 88-1).
Stability of Genotype and Phenotype
The capacity to actively proliferate is often associated with the accumulation of chromosomal abnormalities and mutations. Mouse ES cells appear to be an exception to this rule and tend to maintain their euploid karyotype and genome integrity. By contrast, human ES cells appear to be more susceptible to mutations after long-term culture. However, it is also important to note that even euploid mouse ES cells can form teratomas when injected into immunosuppressed animals, raising concerns about the possible formation of tumors after transplanting actively dividing stem cells.
POTENCY AND DIFFERENTIATION OF STEM CELLS
The term potency is used to indicate a cell’s ability to differentiate into multiple specialized cell types. The current lack of knowledge about the molecular nature of potency requires the experimental manipulation of stem cells to demonstrate their potency. For example, in vivo testing can be done by injecting stem cells into mouse blastocysts or immunosuppressed adult mice and determining how many different cell types are formed from the injected cells. However, these in vivo assays are not applicable to human stem cells. In vitro testing can be performed by differentiating cells in various culture conditions to determine how many different cell types are formed from the cells. The formal test of self-renewal and potency is performed by demonstrating that a single cell possesses such abilities in vitro (clonality). Cultured stem cells are tentatively grouped according to their potency (Fig. 88-1). Only some examples are shown, because many cultured stem cells, especially human cells, lack definitive information about their developmental potency.
Potency and source developmental stage of cultured stem cells. For abbreviations of stem cells, see Table 88-1. Note that stem cells are often abbreviated with or without “cells,” e.g., ES cells or ESCs for embryonic stem cells. h, human; m, mouse.
From Totipotency to Unipotency
Totipotent cells can form an entire organism autonomously. Only a fertilized egg (zygote) possesses this feature. Pluripotent cells (e.g., ES cells) can form almost all of the body’s cell lineages (endoderm, mesoderm, and ectoderm), including germ cells. Multipotent cells (e.g., HS cells) can form multiple cell lineages but cannot form all of the body’s cell lineages. Oligopotent cells (e.g., NS cells) can form more than one cell lineage but are more restricted than multipotent cells. Oligopotent cells are sometimes called progenitor cells or precursor cells; however, these terms are often more strictly used to define partially differentiated or lineage-committed cells (e.g., myeloid progenitor cells) that can divide into different cell types but lack self-renewing capacity. Unipotent cells or monopotent cells (e.g., spermatogonial stem [SS] cells) can form a single differentiated cell lineage.
Development naturally progresses from totipotent fertilized eggs to pluripotent epiblast cells to multipotent cells and, finally, to terminally differentiated cells. According to Waddington’s epigenetic landscape, this is analogous to a ball moving down a slope. The reversal of the terminally differentiated cells to totipotent or pluripotent cells (called nuclear reprogramming) can thus be seen as an uphill gradient. Nuclear reprogramming has been achieved using nuclear transplantation, or nuclear transfer (NT), procedures (often called “cloning”), where the nucleus of a differentiated cell is transferred into an enucleated oocyte. Although this is an error-prone procedure with a very low success rate, live animals have been produced using adult somatic cells as donors in sheep, mice, and other mammals. In mice, it has been demonstrated that ES cells derived from blastocysts made by somatic cell NT are indistinguishable from normal ES cells. NT can potentially be used to produce patient-specific ES cells carrying a genome identical to that of the patient, although such strategies have not been pursued due to ethical issues and technical challenges. Recent success in generating human ES cells by NT has rekindled an interest in this area; however, the limited supply of human oocytes will still be a major problem for clinical applications of NT.
An alternative approach that has become a method of choice is the direct conversion of terminally differentiated cells into ES-like cells (called induced pluripotent stem [iPS] cells) by overexpressing a combination of key transcription factors (TFs). The original method was to infect mouse embryonic fibroblast cells with retrovirus vectors carrying four TFs [Pou5f1 (Oct4), Sox2, Klf4, and Myc] and to identify rare ES-like cells in culture. This approach was soon adapted to human cells, followed by a more refined procedure (e.g., the use of fewer TFs, different cell types, and different gene-delivery methods). Because a clinical trial using iPS cells is imminent, the safety of iPS-based therapy is a major concern and a variety of measures are being taken to ensure the safety. For example, it has now become a standard to use footprint-free methods such as an episomal vector, Sendai virus vector, and synthetic mRNAs to deliver reprogramming factors into cells, resulting in the production of patient-specific iPS cells with minimal alteration of their genetic makeup. In addition to cell replacement therapy, disease-specific iPS cells are expected to play a role in modeling human disease in vitro and in screening drugs for personalized medicine.
It has also become possible to convert one type of terminally differentiated cell (e.g., fibroblast cell) into another type of terminally differentiated cell (e.g., cardiac muscle, neuron, or hepatocyte) by overexpressing specific sets of TFs (called direct reprogramming). Direct reprogramming can bypass the step of making iPS cells, possibly providing the safer route to desired cell types for therapy; however, the technology is currently limited by its low efficiency.
Stem Cell Plasticity, Transdifferentiation, and Facultative Stem Cells
The prevailing paradigm in developmental biology is that once cells are differentiated, their phenotypes are stable. However, more recent studies show that tissue stem cells, which have traditionally been thought to be lineage-committed multipotent cells, possess the capacity to differentiate into cell types outside their lineage restrictions (called transdifferentiation). For example, HS cells may be converted into neurons as well as germ cells. This feature may provide a means to use tissue stem cells derived directly from a patient for therapeutic purposes, thereby eliminating the need to use embryonic stem cells or elaborate procedures such as nuclear reprogramming of a patient’s somatic cells. However, more strict criteria and rigorous validation are required to establish tissue stem cell plasticity. For example, observations of transdifferentiation may reflect cell fusion, contamination with progenitor cells from other cell lineages, or persistence of pluripotent embryonic cells in adult organs. Therefore, the assignment of potency to each cultured stem cell in Fig. 88-1 should be considered with caution. Whether transdifferentiation exists and can be used for therapeutic purposes remains to be determined conclusively. A similar, but distinct, concept is the facultative stem cell, which is defined as a unipotent cell or a terminally differentiated cell that can function as a stem cell upon tissue injury. The presence of such cells has been proposed for some organs such as liver, intestine, pancreas, and testis, but is still debated.
Directed Differentiation of Stem Cells
Pluripotent stem cells (e.g., ES and iPS cells) can differentiate into multiple cell types, but in culture, they normally differentiate into heterogeneous cell populations in a stochastic manner. However, for therapeutic uses, it is desirable to direct stem cells into specific cell types (e.g., insulin-secreting beta cells). This is an active area of stem cell research, and protocols are being developed to achieve this goal. In any of these directed cell differentiation systems, the cell phenotype must be evaluated critically. Alternatively, the heterogeneity of the cell population derived from pluripotent stem cells can be actively exploited, as different types of cells interact with each other in culture and further enhance their own differentiation. In some instances, e.g., optic cup, self-organizing tissue morphogenesis has been demonstrated in 3D culture.
MOLECULAR CHARACTERIZATION OF STEM CELLS
In addition to standard molecular biological approaches, high-throughput genomics and proteomics have been extensively applied to the analysis of stem cells. For example, DNA microarray analyses have revealed the expression levels of essentially all genes and identified specific markers for some stem cells. Chromatin immunoprecipitation coupled with next-generation sequencing technologies, capable of producing billions of sequence reads in a single run, has revealed chromatin modifications (“epigenetic marks”) relevant to stem cell properties. Similarly, the protein profiles of stem cells have been assessed by using mass spectrometry. These methods are beginning to provide a novel means to characterize and classify various stem cells and the molecular mechanisms that give them their unique characteristics.
It is important to identify genes involved in the regulation of stem cell function and to examine the effects of altered gene expression on ES and other stem cells. For example, core networks of TFs such as Pou5f1 (Oct4), Nanog, and Sox2, govern key gene regulatory pathways/networks for the maintenance of self-renewal and pluripotency of mouse and human ES cells. These TF networks are modulated by specific external factors through signal transduction pathways, such as leukemia inhibitory factor (Lif)/Stat3, mitogen-activated protein kinase 1/3 (Mapk1/3), the transforming growth factor β (TGFβ) superfamily, and Wnt/glycogen synthase kinase 3 beta (Gsk3b). Inhibitors of Mapk1/3 and Gsk3b signaling enhance the derivation of ES cells and help maintain ES cells in full pluripotency (“ground” or “naive state”). Recent data also indicate that 20–25 nucleotide RNAs, called microRNAs (miRNAs), play an important role in regulating stem cell function by repressing the translation of their target genes. For example, it has been shown that miR-21 regulates cell cycle progression in ES cells and miR-128 prevents the differentiation of hematopoietic progenitor cells. These types of analyses should provide molecular clues about the function of stem cells and lead to a more effective means to manipulate stem cells for future therapeutic use.