Stem Cells

Stem Cells 101
How these powerful entities operate and some recent developments in stem cell biology


REVIEW SUMMARY

1. Stem cells (SCs) are divided into two main types:


Embryonic stem cells derive from the blastocyst and can, in theory, give rise to all cell types in an organism.

Adult stem cells derive from mature tissue and usually have a restricted spectrum of possible differentiation (e.g., epidermal stem cells).


2. Stem cells have two cardinal features:


The ability to self-renew indefinitely through cell division

The ability to differentiate into varying cell types


3. Biologists have begun to unravel the genetic determinants that dictate "stemness."

4. Normal skin fibroblasts can be "reprogrammed" into a stem-cell–like phenotype (induced pluriopotent stem cells) through the introduction of a limited number of genes.

5. Stem cell therapy is theoretically ideal for degenerative human disorders; proof-of-principle has already been established in mice models of disease.

Stem cells have been a source of tremendous promise and controversy during the past decade. Some contention derives from conflicts between secular and religious definitions of life. Another source of controversy is the imprecise use of stem cell terminology and concepts. The purpose of this brief review is to clarify principles and showcase some recent advances in SC biology.

FUNCTIONAL ATTRIBUTES

SCs have traditionally been defined by their functional attributes. There are two cardinal features of all SCs. The first is self-renewal — the capacity to go through indefinite cycles of cell division while maintaining an undifferentiated state. To self-renew, SCs undergo either symmetric division or asymmetric division. In symmetric division, a stem cell gives rise to two identical daughter cells, both endowed with SC properties:

A A + A*
In asymmetric division, an SC gives rise to one stem cell and one progenitor cell with limited self-renewal potential:

A A + B*
Progenitor cells have the capacity to undergo several cycles of cell division,

B B + B*
but unlike stem cells, will eventually differentiate terminally.

B C + C*
(*A = adult stem cell; B = progenitor cell; C = terminal, differentiated cell)

The mechanisms that govern the activation of symmetric versus asymmetric division and that determine which daughter cells will be stem cells and which will be progenitor cells are still unclear.

The second cardinal feature of all SCs is potency — the ability to differentiate into specialized cell types. There are four major types of potency:


Totipotency is the ability of a single cell to expand in number, differentiate into embryonic and extraembryonic tissues, and develop into an organism.

Pluripotency is the ability to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm.

Multipotency is the capacity to produce a related family of cells (e.g., hematopoietic SCs differentiate into various blood cell types).

Unipotency is the ability to differentiate into just one cell type (e.g., epidermal SCs generate only keratinocytes). Different sources and types of SCs may harbor different types of potency.


STEM CELL CLASSES
The two large classes of SCs are embryonic stem cells (ESCs), which are found in developing blastocysts, and adult stem cells (ASCs), which are found in mature tissues. ESCs derive from fertilized embryos; specifically, they are isolated from the epiblast tissue of the inner cell mass of a blastocyst or earlier morula. In humans, a blastocyst comprises about 50 to 150 cells and is approximately 4 to 5 days old. ESCs conform to the definition of pluripotency, in that they can develop as all three primary germ layers: ectoderm, endoderm, and mesoderm. ESCs do not contribute to the extraembryonic membranes or the placenta. Although there are phenotypic similarities between mouse and human ESCs, each requires very distinct experimental conditions for study.

ASCs can be found in the developed organism in both children and adults. ASCs are also known as somatic SCs and are often lineage-restricted (i.e., multipotent but not pluripotent). In the fully formed organism, ASCs are conventionally described according to the tissue of origin (e.g., epidermal SCs, adipose SCs, mesenchymal SCs). For instance, a certain type of ASC, the hematopoietic stem cell (HSC), has been used for decades in the treatment of certain bloodborne cancers. Bone marrow transplantation is successful because HSCs transfer and engraft. Michele De Luca and colleagues recently applied SC technology to treatment of junctional epidermolysis bullosa (JW Dermatol Jan 26 2007 and Nat Med 2006; 12:1397). From palmar skin, they isolated putative SCs, into which they introduced the defective laminin 5 (LAM5) subunit and used the engineered cells to prepare genetically corrected cultured epidermal grafts. The autologous grafts showed synthesis and proper assembly of normal levels of functional LAM5. Even after a year, the engineered epidermis remained stable and adherent without blisters, infections, inflammation, or immune response. Although longer-term follow-up with more patients is needed to validate this study, the use of ASCs for certain targeted tissues is appealing, as it does not involve embryos.

IDENTIFYING STEM CELLS AND STEM CELL UTILITY
The identification and isolation of SCs has been challenging. No single fixed criterion offers indisputable evidence of "stemness." The sine qua non of an SC is its ability to regenerate a certain tissue over the life of the organism; thus, SCs are particularly attractive therapeutic models for degenerative diseases, such as neuronal degeneration in Parkinson disease or pancreatic degeneration in diabetes. An extant example of SC utility is the reconstitution of a complete immune system from bone marrow, which, presumably, contains hematopoietic SCs. In vitro assays help us understand the cellular behavior of SCs. For example, in the well-established clonogenic assay, SCs are characterized by their capacity to form colonies in tissue culture plates when seeded at low density. By contrast, committed cells that have limited replicative or survival potential will not undergo enough rounds of cell division to form clonal colonies. More-recent work has attempted to identify SCs based on protein or RNA markers. For instance, ESCs have been isolated and found to express transcription factors that appear to maintain pluripotency (Cell 2005; 122:947). The ability to point to the master regulators of "stemness" has been leveraged to create a new generation of induced SCs from adult tissue.

STEM ALCHEMY: INDUCED PLURIPOTENT STEM CELLS
In the Middle Ages, the art of transforming common metals into gold became a source of great mystical enthusiasm (and ultimate disappointment). Given the controversial nature and rarity of ESCs, the transformation of somatic cells into ESCs has similarly engendered great excitement in the past few years. Unlike the alchemists, however, current researchers have had some successes in SC engineering that may eventually find a therapeutic outlet.

The theoretical framework for production of induced pluripotent stem cells (iPSCs) is relatively straightforward: Identify a set of genes that define the "stemness" of ESCs and then "reprogram" adult cells as ESC-like cells by introducing these genetic factors into the somatic cells. Induced PSCs were first generated in mice in 2006 (Cell 2006; 126:663). Initial observations showed that four key genes were essential for reprogramming differentiated cells into the pluripotent state: Oct-3/4, Sox2, c-Myc, and Klf4. Despite similarities with ESCs, early iPSC lines failed to produce viable chimeras when injected into mouse embryos; thus, they do not fulfill the in vivo criteria for ESCs. Chimeras are animals that develop from different genetic sources — in this case, animals derived from both the host and the iPSCs. Subsequent refinements in technique have led to the successful reprogramming of mouse fibroblasts into iPSCs that can, in fact, produce viable chimeras (Nature 2007; 448:313 and Nature 2007; 448:318), thereby fully substantiating their pluripotent nature.

Induced PSCs have been shown to be similar to ESCs in morphology, growth properties, and the expression of SC genes and SC markers. Most critically, iPSCs can be triggered to differentiate into cardiac and neural cells as well as form viable mouse chimeras. A major concern among iPSC investigators is the use of c-Myc — a known oncogene — in the reprogramming; in fact, a significant fraction of the derived mice later developed cancer. A more recent protocol may make it possible to dispense with c-Myc (Nat Biotechnol 2008; 26:101).

Figure 1: Skin fibroblasts as the source of pluripotent human stem cells of the future
Reprinted with permission from Macmillan Publishers Ltd: Nature Medicine copyright 2007.

Differentiated cells, such as skin fibroblasts, are isolated and genetically reprogrammed to become iPSCs via the introduction of certain genes such as POU5F1, MYC, KLF4, and SOX2. These genes in turn induce the expression of SC master regulators, POU5F1 and NANOG. The iPSCs can then be recovered and redifferentiated into therapeutically regenerative cells and tissue. For details, see Nat Med 2007; 13:783.

THERAPEUTIC IMPLICATIONS
So what are the potential therapeutic implications of iPSCs? No human trials of iPSCs are under way, but at MIT, Rudolf Jaenisch and colleagues have recently shown that fibroblasts can be reprogrammed into fully functional blood cells in a humanized mouse model of sickle cell anemia (Science 2007; 318:1917). The same group also showed that iPSCs can be efficiently differentiated into neural precursor cells; transplanted into the fetal mouse brain; and successfully form glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. In addition, when transplanted into the adult brain in a rat model of Parkinson disease, these iPSCs were able to improve behavior (Proc Natl Acad Sci U S A 2008; 105:5856). Studies involving skin disease will undoubtedly be published over the course of the next few years.

The National Institutes of Health provide a good introductory discussion of stem cells and a useful glossary of related terms.

— Hensin Tsao, MD, PhD

Published in Journal Watch Dermatology June 13, 2008