Alternative sources of stem cells

Embryonic stem (ES) cells are totipotent but have been shown to form tumors in immunodeficient mice. ES cells grow as teratocarcinomas in vivo and frequently acquire chromosomal aberrations. They have restricted differentiation capacity and have been shown to yield both genetic and epigenetic abnormalities in culture. Significant ethical issues arise regarding the use of embryos as well.

Cord blood cells are expanded easily but are primarily hematopoietic lineage cells with <1% multipotent cells. There is substantial clinical experience with cord blood transplantation for hematologic indications and growing interest in other applications. Umbilical cord and placental stem cells exist in the middle ground between ES cells and adult stem cells and might yield desirable expansion with lack of tumorigenesis. However, expansion and culture studies are limited at present.

Bone marrow is the most common source of adult stem cells for clinical transplantation. Restricted lineage, low proliferation rates because of limited telomerase expression, and restricted differentiation potential limit their clinical application so far. They are, however, less tumorigenic than ES cells and do not have ethical concerns. There is extensive clinical experience with their use, establishing standard protocols for transplantation.

Amniotic fluid is readily available, is noncontroversial, and is obtained routinely by amniocentesis. It is analyzed for prenatal genetic diagnosis in the second trimester, for evidence of fetal pulmonary maturity in the third trimester, and for infection at any gestational age. It is made up of a heterogeneous population of cells that routinely is retrieved clinically and cultured for genetic studies. It has been shown that a significant percentage of cells that are obtained from amniocentesis exhibit stem-cell markers. These cells are cultured easily, expanded, and remain viable over many passages, tolerating cryopreservation very well.

A great deal of research has been performed on second-trimester amniotic fluid cells (AFC), but there is little known about whether third-trimester AFC closely resemble those of the mid-trimester. You et al retrieved amniotic fluid at the time of elective cesarean delivery at term. AFC were isolated in culture and found to be positive for surface markers CD29, CD73, CD90, and CD105 as well as Oct-4. Proliferation potential was verified, and differentiation into osteogenic lineage was reported as successful. A more recent study aimed to characterize human AFC from the early third trimester, where 3 samples were analyzed from gestations from 28-34 weeks. Third-trimester AFC expressed comparable levels of Oct-4 and Nanog, but lower levels of SOX2 and Rex-1. Samples were also successfully differentiated to adipocytes, osteoblasts, chondroblasts, myocytes, and neural-like cells, although third-trimester samples showed poor differentiation potential to myocytes and stronger potentiation to neural lineage. Further studies on not only third-trimester amniotic fluid but also AFC at term are warranted, because these findings are promising but inconclusive.

AFC

In early studies, amniotic fluid from pregnant ewes was isolated and expanded to mesenchymal, fibroblast/myofibroblast cell lineages. These cells were noted to proliferate significantly faster than surrounding cells, showed little cell death, and could be isolated consistently from amniotic fluid. AFC showed stem-cell potential when they were found to contain Oct-4, which is a known marker specific for human embryonic stem cells that are associated with maintenance of the undifferentiated state and pluripotency. Besides their rapid proliferation, human AFC have been differentiated successfully into all embryonic germ layers, thereby demonstrating pluripotency. After multiple passages in culture, human AFC remained chromosomally stable and did not form teratomas or undergo malignant change. These qualities of human AFC suggested significant advantages as a potential source of cells for clinical transplantation.

ES cell molecular markers are molecules that specifically are expressed by stem cells and are critical to the characterization and identification of their pluripotency. There are a wide range of cell-surface proteins, transcription factors, and molecular markers indicative of stemness ( Table ). These surface markers are usually glycosphingolipids or membrane proteins. Research on stem cells has used the technique of flow cytometry. Flow cytometry separates cells using their cell surface markers, thereby identifying viable cells. This process then permits the formation of clonal cultures from specifically identified cells. Flow cytometry can also be used to identify transcription factors in cell nuclei that are related to stem-cell behavior. However, in contrast to the process of identification of cell surface markers, the cells are no longer viable and cannot be cultured after transcription factor identification.

Human amniotic fluid–derived stem cells were identified as expressing Oct-4, SOX2, Nanog, Rex1, cyclin A, and mesenchymal markers that include CD90, CD105, CD73, CD166, CD133, and CD44. There is a higher percentage of stem-cell transcription factors Oct-4, Nanog, and SOX2 in AFC from fluid that is obtained from 15-17 weeks gestation vs later gestational ages. Cell surface markers do not appear to vary with gestational age among the second-trimester samples, but individual samples’ expression varies greatly and may overshadow this effect. The ability for adipocyte, osteocyte, and neuronal cell generation were also demonstrated. De Coppi et al postulated that CD117 was a marker for selection of AFC from human amniocentesis cultures. However, subsequently, it was shown that CD117 is actually a clonal marker that was present in only approximately 0.5-2% of AFC in cultures that used the same media. Another variety of human AFC, mesenchymal stem cells have been identified with distinct populations of varying differentiation potential.

Our studies on human AFC have shown the presence of CD117, 133, 90, 15, 44, 29, 9, 73, as well as SSEA1, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Oct4, Rex1, Nanog, and SOX2. Our laboratory selected SSEA4, Tra-1-60, and CD90 for subsequent investigations because they were the most highly expressed ES cell markers in our patient samples. These studies have also shown clonal populations bearing all 3 markers, combinations of 2 different stem cell markers (eg, SSEA4/CD90, SSEA4/TRA-1-60, and Tra-1-60/CD90), and populations with just 1 marker. Clones with different combinations of markers may vary in their properties of stemness. This is an area for further investigation that has not yet been explored. Thus, there is a mixture of cells with varying potential for differentiation into lineages from all 3 germ layers, likely in addition to cells that are pluripotent.

As yet, it is not clear which clones will have different potentials for differentiation, but clonal isolation of a single pluripotent clonal cell line is not essential for AFC differentiation. Because of the heterogeneous nature of AFC, it may not be important to isolate a pluripotent stem-cell clone to build a framework with which to move towards translational use in therapy. Our laboratory studied 37 different samples of human amniotic fluid. A total of 81 cultures were obtained for study from doubling each culture sample from 2-8 times. With every culture, there was consistent inducibility to osteogenic, chondrogenic, and neurogenic lineage without the need for isolation of a specific clone with either a single marker or combination of markers. Individual variation in the percentage distribution of cells that express different markers does not appear to be a limitation because every sample has a wide variety of lineage precursors. Amniotic fluid–derived cells have been reprogrammed easily into induced pluripotent stem cells, which supports this concept. This is in contrast to the concerns raised by Ekblad et al. They focused on selecting a single CD117-positive pluripotent cell as proposed by Zia et al and were unsuccessful in enriching the CD117 population. Thus, it is likely that clonal selection is unnecessary for clinical use.

AFC

In early studies, amniotic fluid from pregnant ewes was isolated and expanded to mesenchymal, fibroblast/myofibroblast cell lineages. These cells were noted to proliferate significantly faster than surrounding cells, showed little cell death, and could be isolated consistently from amniotic fluid. AFC showed stem-cell potential when they were found to contain Oct-4, which is a known marker specific for human embryonic stem cells that are associated with maintenance of the undifferentiated state and pluripotency. Besides their rapid proliferation, human AFC have been differentiated successfully into all embryonic germ layers, thereby demonstrating pluripotency. After multiple passages in culture, human AFC remained chromosomally stable and did not form teratomas or undergo malignant change. These qualities of human AFC suggested significant advantages as a potential source of cells for clinical transplantation.

ES cell molecular markers are molecules that specifically are expressed by stem cells and are critical to the characterization and identification of their pluripotency. There are a wide range of cell-surface proteins, transcription factors, and molecular markers indicative of stemness ( Table ). These surface markers are usually glycosphingolipids or membrane proteins. Research on stem cells has used the technique of flow cytometry. Flow cytometry separates cells using their cell surface markers, thereby identifying viable cells. This process then permits the formation of clonal cultures from specifically identified cells. Flow cytometry can also be used to identify transcription factors in cell nuclei that are related to stem-cell behavior. However, in contrast to the process of identification of cell surface markers, the cells are no longer viable and cannot be cultured after transcription factor identification.

Table

Human embryonic stem-cell markers

Marker	Characteristic	Classification	Known activity
Stage-specific embryonic antigens (cell surface associated)
SSEA-1 (CD15/Lewis x)	EC, ES	Carbohydrate-associated; lactoseries oligosaccharide antigen	Increase with differentiation
SSEA-3 (glycolipid GB5)	EC, ES, EG	Carbohydrate-associated; globoseries carbohydrate antigen	Decrease with differentiation
SSEA-4	EC, ES, EG	Carbohydrate-associated glycoprotein	Decrease with differentiation
Transcription factors
Nanog	ES	Homeobox family, DNA-binding transcription factor	Decrease with differentiation
Oct-4 (POU5F1)	EC, ES, EG	Octamer-binding transcription factor	Most recognized marker totipotent ES
Rex-1 (Zfp42)	EC, ES	Zinc finger family	Maker of undifferentiated cells
SOX2	EC, ES	SOX family, DNA-binding transcription factor	Specifies germ lineage at implantation, regulates proliferation, differentiation
TRA-1-60	EC, ES, EG	Surface antigen, epitope expressed on high molecular weight proteoglycan	Tumor recombinant antigen
TRA-1-81	EC, ES, EG	Surface antigen, epitope expressed on podocalyxin	Tumor recombinant antigen
Cluster of differentiation markers
CD9 (MRP1)	ES	Surface marker, tetraspanin family	Cell adhesion, migration, T-cell stimulation
CD29 (B1 integrin)	ES	Surface marker, intregrin family	Cell adhesion, recognition, embryogenesis
CD44	EC, ES	Surface marker, glycoprotein	Cell interactions, adhesion, migration
CD45 (PTPRC)	ES	Protein tyrosine phosphatase receptor; leukocyte common antigen	Cell growth and differentiation, mesenchymal, hematopoetic cells
CD73	ES	Surface marker, enzyme	Converts adenosine monophosphate to adenosine
CD90 (Thy-1)	EC, ES	Surface marker, GPI-linked glycoprotein	Mesenchymal stromal cells, hematopoietic stem cell, neuron, T-cell activation
CD105	ES	Surface marker, endoglin, transmembrane glycoprotein of zona pellucida	Differentiation of smooth muscle, angiogenesis, neovascularization
CD117 (c-kit)	EC, ES	Surface marker, stem-cell factor receptor, tyrosine kinase receptor	Present on hematopoietic progenitor cells, role in gametogenesis
CD133	EC, ES	Surface marker, Prominin-1, glycoprotein	Organizer of cell membrane topology
CD166	EC, ES	Surface marker, immunoglobulin	Cell interaction and adhesion

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