Stem Cell Reviews and Reports

, Volume 7, Issue 4, pp 782–796 | Cite as

A Comparison of Stem Cells for Therapeutic Use



A critical comparison of the attributes of several types of stem cells is presented, with particular emphasis on properties that are critical for the application of these cells for therapeutic purposes. The importance of an autologous source of pluripotent stem cells is stressed. It is apparent that two sources currently exist for non-embryonic pluripotent stem cells—very small embryonic-like stem cells (VSELs) and induced pluripotent stem cells (iPS). The impact of the emerging iPS research on therapy is considered.


Pluripotency Multipotency Embryonic stem cells Induced pluripotent stem cells Adult stem cells Very small embryonic-like stem cells Autologous stem cells Regenerative medicine 


Considerable confusion exists with respect to the capabilities, attributes, and deficiencies of adult stem cells. Initially this term was used to distinguish stem cells (including cells derived from non-adult material such as cord blood) from embryonic stem cells obtained from the inner mass (embryoblast) of the blastocyst. While little doubt exists about the importance of embryonic stem cells as a tool to understand mechanisms of cell differentiation and other cellular mechanisms, it is generally accepted that embryonic stem cells will have a limited place in the development of therapies until the problems associated with these cells (inherent tumorigenesis, graft-versus-host disease, and other issues precipitating regulatory interventions) are resolved [1, 2]. Clearly, embryonic stem cells can never be autologous and, thus, are deprived of a major attribute achievable with adult stem cells. In addition, while much of the regulatory restriction on embryonic stem cell research has been removed by the current U.S. Administration, considerable concern about ethical issues remains [3].

Stem cells are critical to embryo development and are retained through fetal growth into adulthood as a homeostatic mechanism for repair of injured tissue and replacement of senescent cells through regeneration [4]. During this progression, stem cells, in addition to tissue generation, migrate through the fetal circulation and home to a number of locations including, preferentially, the bone marrow. This migration is both active and gradient driven (e.g., the stromal-derived factor-1 (SDF-1) gradient for hematopoietic locations such as the bone marrow and fetal liver) as well as passive, (e.g., the trapping of stem cells in the various organ stem cell niches), or a combination of active and passive processes [5, 6]. The stem cells contained in these niches respond to stimuli generated by injury, by differentiating into the required cell type, and by self-renewal, thereby maintaining a reservoir of stem cells for future differentiation and deployment [7, 8]. In addition, presumably initiated by the same stimuli and mediated by signaling molecules, stem cells are mobilized from the bone marrow and migrate to the site of injury; there, they are recruited and integrated into that tissue to perform the required functions of regeneration through transdiffentiation, or through paracrine, trophic, or other processes.

Adult stem cells are now the basis of essentially all successful stem-cell based therapies. By definition, an adult stem cell is an undifferentiated cell found among differentiated cells; in a tissue or organ, it can renew itself and differentiate, through progenitor cells, to yield the major specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. Some now prefer the use of the term somatic stem cell instead of adult stem cell. One population, called hematopoietic stem cells or CD34+ stem cells, forms all of the blood cell types in the body [9]. A second population, called bone marrow stromal cells, was discovered a few years after hematopoietic stem cells [10, 11]. Stromal cells, also called mesenchymal stem cells, are a poorly-defined mixed cell population that can generate bone, cartilage, fat, and fibrous connective tissue when appropriately stimulated and expanded in vitro (Table 1). Mesenchymal stem cells were once believed to be a universal solution to tissue regeneration, but current thinking suggests that these cells have greater importance as immune modulators, with their regenerative properties being due to trophic paracrine effects rather than true regeneration [12]. Hence a more appropriate name for these cells would be mesenchymal stromal cells.
Table 1

Key characteristics of adult multipotent stem cells and very small embryonic-like stem cells

Adult multipotent stem cells

 • Undifferentiated stem cell existing within a mature organ/tissue

      ◦ Can be a source of new stem cells or

      ◦ Can act to renew/repair damaged cells within that organ/tissue

 • Multiple sources: bone marrow, peripheral blood, adipose tissue, cord blood and placenta, teeth, menses

 • Previously thought to be able to differentiate into a limited number (multipotent) of cell types only

 • Adult stem cells (e.g., MSCs) permit patients to act as their own donor (autologous cell-based therapy), with no risk of rejection and quicker engraftment (in contrast to hESC)

VSEL pluripotent adult stem cells

 • Stem cells that have the potential of true pluripotency (can differentiate into nearly all cells) without the drawbacks of their embryonic counterparts

 • Derived from peripheral blood (no need for bone marrow aspiration); no human embryo destroyed

 • Small numbers of VSELs can provide adequate cell doses and are expandable (in animal studies)

 • Cryopreservation is possible to provide a lifelong deposit of pluripotent stem cells

 • VSELs enable autologous (patients’ own) treatment:

      ◦ No risk of rejection or graft vs. host disease

      ◦ No risk of infectious diseases

      ◦ No tissue match requirement so available when needed

      ◦ Potentially faster recovery times with functionality

 • Numerous therapeutic applications: non-healing wounds, severe burns, eye injuries in troops

hESC human embryonic stem cells; MSCs mesenchymal stem cells; VSEL Very Small Embryonic-Like

Confusion also exists with respect to the use of potency as a descriptor of the range of differentiating capabilities of stem cells. In particular, considerable overlap exists in the use of the terms multipotent and pluripotent. In large part, this has arisen because of the somewhat restrictive requirements imposed by early workers to define pluripotency in cells obtained from embryos (Table 2). The definition of pluripotency for adult stem cells should be restricted to the ability of a stem cell to differentiate into all three germ layers, since the criteria applied to embryonic stem cells (teratoma formation and germ-line transfer to blastocysts), have no bearing on the therapeutic potential of adult stem cells. The ability of a cell to transdifferentiate into cells of all three germ layers will, in addition, have a requirement for “primitivism” as reflected by the expression of genetic markers (transcription factors) and cell surface antigenic markers such as Oct4, Nanog, stage-specific embryonic antigen (SSEA), etc. The necessity of having the teratoma formation ability as a condition of pluripotency would be considered a strong negative with respect to the assessment of therapeutic potential.
Table 2

Criteria for stem cell pluripotency based on embryonic stem cells


Mouse embryonic stem cells (mES Cells)

Human embryonic stem cells (hES Cells)

Induced pluripotent stem cells (iPS)



Very small embryonic-like stem cells (mVSEL)

Very small embryonic-like stem cells (hVSEL)

Expression of genetic markers (Oct4, Nanog, etc.)






Differentiation into cells of all three germ layers





As yet unknowna

Formation of teratomas with cells of all germ layers






Germ-line transfer following blastocyst injection






aNeoStem Research Laboratory, Cambridge, MA, is currently evaluating the tri-lineage potential of hVSELs

bVSELs were shown to have a unique genetic imprinting, which supports their quiescent (non-proliferative) state, unlike ES cells which readily form teratomas [13]

cBlastocyst injections were not attempted with VSELs. Due to the unique methylation status of the imprinted genes in VSELs, they are unlikely to contribute to germ-line transfer unless the proper methylation status is restored

Very-small Embryonic-like Stem Cells

Recent evidence suggests that bone marrow contains a heterogeneous population of non-hematopoietic stem cells, in addition to well-described hematopoietic stem cells [14, 15]. Furthermore, many researchers have postulated that these bone marrow, non-hematopoietic stem cells contain some very rare cell populations that display several cardinal features of pluripotent stem cells [16, 17]. These stem cells are capable of differentiating into cells of all three germ layers and hence can be considered to be pluripotent. Among these cells are the stem cells described by Friedenstein and by Prockop [18, 19, 20]; the multipotent adult progenitor cells described by Verfaillie and her colleagues [21]; the so-called MIAMI (marrow-isolated adult multilineage inducible) cells by Schiller et al. [22, 23]; the multipotent adult stem cells described by Beltrami et al. [24]; and very small embryonic-like stem cells (VSELs), which are so called since they are smaller than mononuclear cells, with a size ranging from approximately 3 microns (murine) to 7 microns (human), as described by Kucia et al. [14, 25].

It is possible that all of these primitive stem cells, which have great therapeutic potential, are similar; unfortunately, not all have been well characterized on a cellular and functional basis. Thus, the potential relationships among the above cells are not clear. It is possible that these cells are overlapping populations of primitive stem cells in human bone marrow and human cord blood identified by various isolation and expansion strategies, but assigned different names [26]. Furthermore, none of these cells had been isolated at the single-cell level before Ratajczak and his group succeeded to do so for VSELs [27, 28]. Now, in the case of murine and, to a lesser extent, human VSELs, a clearer picture is emerging of their properties, purification, origin, and functional capabilities.

Isolation of VSELs

Murine VSELs were initially isolated from bone marrow by employing multiparameter cell sorting and the use of a novel size-based approach using size bead markers to extend the lymphocyte gate to include the so-called small event region (2–10 microns). While this region mostly contains cell debris, large platelets, and erythrocytes, it also includes some rare nucleated cells. Most cell sorting protocols exclude events smaller than 6 microns in diameter to avoid cell debris, erythrocytes, and platelets; therefore, small VSELs are usually excluded from the sorted cell populations and, as a consequence, these small cells previously escaped detection.

This flow cytometer gating technique generates a population of rare cells (<0.02% of the total mononuclear cells of the bone marrow), which have CXCR4+Sca-1+ linCD45 cell surface markers and express (as determined by real time polymerase chain reaction [PCR] techniques and immuno-histochemistry) markers of primitive pluripotent stem cells, including SSEA-1, Octamer-4, Nanog, and Rex-1 and Rif-1 telomerase proteins [14]. When examined by direct electron microscopy, these cells show several features typical of embryonic stem cells, such as small size (approximately 3.6 microns in diameter), a large nucleus surrounded by a narrow rim of cytoplasm, and open-type chromatin (euchromatin) (Fig. 1) [29].
Fig. 1

At the ultrastructural level, the narrow rim of cytoplasm possesses a few mitochondria, scattered ribosomes, small profiles of endoplasmic reticulum, and a few vesicles. The nucleus is contained within a nuclear envelope with nuclear pores. Chromatin is loosely packed and consists of euchromatin. These characteristics are highly comparable to undifferentiated embryonic stem cells. Reproduced with permission from Kucia et al. [29]

VSELs have been purified from mouse bone marrow by the process outlined above in Fig. 2 [14]. Despite their small size, VSELs possess diploid DNA. When these purified VSELs are cultured by placing them on a C2C12 murine sarcoma cell feeder layer, they form spheres that resemble embryoid bodies [27]. Cells from these VSEL-derived spheres are composed of immature cells with large nuclei that contain euchromatin and, like purified VSELs, are CXCR4+SSEA-1+Oct-4+. Furthermore, cells from VSEL-derived spheres, after re-plating over C2C12 cells, may again (up to five to seven passages) grow new spheres or, if plated into cultures that promote tissue differentiation, expand into cells of all three germ-cell layers. However, the formation of these spheres appears to be highly dependent on the age of the mouse since no spheres are formed from VSELs derived from mice older than approximately 2 years. Similar spheres were also formed by VSELs isolated from murine fetal liver, spleen, and thymus, substantiating the concept that VSELs are pluripotent and, very importantly, can be expanded in culture. The pluripotency of murine VSELs has been established independently by Taichman et al. at the University of Michigan [30].
Fig. 2

a Bone marrow mononuclear cells freshly isolated from bone marrow are loaded into the upper chamber of transwells and allowed to undergo chemotaxis for 1–3 h to an stromal-derived factor-1 (SDF-1) gradient or control medium (Step I). b Cells that responded to the SDF-1 gradient (expressing functional CXCR4) were subsequently sorted by fluorescence activated cell sorting (FACS) analysis into populations of CD45+ and CD45 cells (Step II). CXCR4+CD45 cells, in contrast to CXCR4+CD45+, are highly enriched in non-hematopoietic developmental markers, including markers of VSEL cells. Reproduced with permission from Kucia et al. [14]

VSELs in Murine Peripheral Blood

Ratajczak and co-workers have provided further evidence that, in steady state conditions, VSELs circulate at very low levels in peripheral blood (approximately 100–200 cells/mL) [31]. Additionally, these cells can be mobilized from the bone marrow to the peripheral blood by granulocyte-colony stimulating factor (G-CSF)-induced or stress-related mobilization, as demonstrated in a mouse model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively.

Other stress-inducing situations can also cause the mobilization of VSELs into the peripheral blood. For example, it has been shown that in the murine model of stroke, a population of primitive cells related to VSELs was mobilized into the peripheral blood [32]. Similarly, in a mouse model for acute myocardial infarction, VSELs were barely detectable in peripheral blood under baseline conditions but their levels increased significantly at 48 h after myocardial infarction, both in younger (6-week-old) and older (15-week-old) mice [33].

If, as suggested by these studies, VSELs play a central role in regeneration, then this should be verifiable in an animal model. Bolli and associates at the University of Louisville School of Medicine, performed a study in mice to examine whether bone marrow derived VSELs promoted myocardial repair after an induced reperfused myocardial infarction [34, 35]. The mice were subjected to a 30-minute coronary occlusion followed by reperfusion. Forty eight hours later, they received a series of five intra-myocardial injections each of 1 × 104 VSELs labeled with enhanced green fluorescent protein (EGFP) injected around the border between infarcted and noninfarcted myocardium. A control group of mice were treated identically but, instead of the VSELs, received an injection of cells of a similar size that were CD45 positive. In addition, a group of mice were treated with the cell-free vehicle only. After 35 days, the VSEL-treated mice showed improved global and regional left ventricle systolic function compared to both non-VSEL treated groups (Fig. 3) [33, 34]. In addition, the VSEL-treated mice showed less left ventricle remodeling and reduced myocardiocyte hypertrophy (Figs. 4 and 5) [33, 34].
Fig. 3

The VSEL-treated group had a smaller left ventricle cavity, thicker infarct wall, and improved motion of the infarct wall. VSEL infusion improved echocardiographic measurements of left ventricle function 35 days after myocardial infarction. All data are compared to values at 96 h. Reproduced with permission from Dawn et al. [34]

Fig. 4

Masson’s trichrome stained myocardial sections from A Vehicle-treated group, B CD45+-treated group, and C VSEL-treated group. Scar tissue is stained blue and viable myocardium is stained red. Reproduced with permission from Dawn et al. [34]

Fig. 5

Assessment of cardiomyocyte and left ventricular hypertrophy in mice after myocardial infarction. Masson’s trichrome stained myocardial sections from A Vehicle-treated group, B CD45+-treated group, and C VSEL-treated group. Scar tissue is stained blue and viable myocardium is stained red. Reproduced with permission from Dawn et al. [34]

These studies suggest that VSELs may play an important role in tissue/organ regeneration in response to injury. Furthermore, VSELs may prove to have all of the potential of embryonic stem cells for the generation of all cell types, but, for reasons explained below, do not have the problem of tumorigenesis that is so frequently associated with embryonic stem cells. In addition, they raise the concept that a primitive pluripotent cell population could be obtained for autologous use in humans, a property impossible to achieve for embryonic stem cells. This finding is of great importance since, for the first time, cells with clearly defined characteristics and a clear propensity to transdifferentiate with high plasticity can be used for autologous cell based therapy. Additionally, no moral, ethical, or religious constraints and federal funding restrictions are imposed on the use of VSELs.

VSELs in Murine Adult Bone Marrow and Organs

It is hypothesized that pluripotent VSELs found in adult bone marrow (and in most other organs of the body) are sequestered in stem cell niches and originate as descendants of epiblast-derived stem cells and primordial germ cells [29, 36]. It is postulated that VSELs represent a dormant and quiescent population of primordial stem cells, a specialized population of primitive stem cells actively contributing to long term regeneration (e.g., hematopoiesis through monopotent cells) and participating in tissue, and possibly organ, regeneration (after mobilization into peripheral blood after stress). At the present time, the most important difference between VSELs and embryonic stem cells is the ability of the latter to form multiple organs and tissues in vivo after injection into a developing blastocyst. This finding is now well accepted with embryonic stem cells isolated from embryos or from embryonic stem cell lines established in vitro [37, 38, 39]. However, to date, this property has been difficult to achieve reproducibly with VSELs [40]. A possible reason for this failure is that VSELs, while retaining most of the properties of embryonic stem cells, have been subjected to the erasure of their somatic imprint.

Ratajczak and colleagues [13] have proposed that VSELs freshly isolated from murine bone marrow erase the paternally methylated imprints (e.g., Igf2-H19, Rasgrf1 loci); however, they hypermethylate the maternally methylated ones (e.g., Igf2 receptor [Igf2R], Kcnq1-p57KIP2, Peg1 loci). According to the “parental conflict theory”, paternally expressed imprinted-genes (Igf2, Rasgrf1) enhance the embryo growth and maternally expressed genes (H19, p57KIP2, Igf2R) inhibit cell proliferation, the unique genomic imprinting pattern observed on VSELs demonstrates growth-repressive imprints in these cells. VSELs highly express growth-repressive genes (H19, p57KIP2, Igf2R) and downregulate growth-promoting ones (Igf2, Rasgrf1), which explains the quiescent status of VSELs [13].

This hypothesis derives from the fact that primordial germ cells initially have a degree of pluripotency comparable to embryonic stem cells, but lose that aspect of pluripotency, together with other competencies, about 9–11 days post-conception [41]. The explanation for this loss of pluripotency in primordial stem cells is dependent on the maintenance of the methylation status of genomic imprinted genes (those genes that are expressed in a parent-of-origin-specific manner). Demethylation of these genes causes a loss of pluripotency and some other capabilities of the primordial stem cell [39, 41]. The methylation pattern necessary for pluripotency can be restored as, for example, in the transition of the primordial stem cell to a sperm or oocyte [39, 42, 43].

Regulation of Murine VSELs Similar to Primordial Stem Cells

To test the hypothesis that VSELs are regulated in a manner similar to primordial stem cells, the methylation status of the Oct-4 promoter and the insulin-like growth factor 2 (Igf2)-H19 locus (an imprinted gene), which is crucial for controlling stem cell pluripotency, in a variety of stem cells including VSELs, were compared using different methods (Fig. 6) [13]. The quiescence of these cells is epigenetically regulated by DNA methylation of genomic imprinting, which is an epigenetic program that ensures the parent-specific mono-allelic transcription of imprinted genes [13]. It has been known for many years that imprinted genes play a crucial role in embryogenesis, fetal growth, totipotential status of the zygote, and most importantly, the pluripotency of early development stem cells [44].
Fig. 6

The DNA methylation patterns of the paternally and maternally imprinted genes of VSELs as compared to hematopoietic stem cells (HSC) and bone marrow derived mononuclear cells (BMNC). Personal communication reproduced with permission from Mariusz Z. Ratajczak, Department of Medicine, University of Louisville, Kentucky

These methylation studies have provided evidence that VSELs show a methylation pattern similar, but not identical, to that of primordial germ cells and supplied experimental support for the concept of their developmental origin being directly from the epiblast/germ line. Ratajczak et al. suggest that these cells are deposited during embryogenesis in tissues with active migration to bone marrow driven by SDF-1 chemotaxis and passive deposition in other organs. The VSELs then serve as backups for tissue-committed stem cells with their proliferative potential tightly regulated by the differential methylation of the Oct-4 promoter, insulin-like growth factor-2-H19 locus, and other maternally and paternally inherited genes. Erasure of methylation at these loci prevents the VSELs from uncontrolled proliferation and from the formation of teratomas. Re-establishment of the methylation pattern of VSELs through the stimulus of appropriate cell signaling processes and/or paracrine effects arising from stress, would bring about reinstitution of their pluripotentiality in vivo, but rapid progression of the VSELs to a differentiated state would preclude tumorigenesis. Graphical representation of this process is shown in Fig. 7 [40].
Fig. 7

Hypothesis for the origin and development of VSELs. The germ line (shown in red) carries the genome (nuclear and mitochondrial DNA) from one generation to the next. All somatic cell lines bud out (gray color) during ontogenesis from the germ line to help germ cells accomplish this mission effectively. The germ potential is established in the fertilized oocyte (zygote), and subsequently retained in the morula, inner cell mass of blastocyst (ICM), epiblast stem cells (EPSC), primordial germ cells (PGC), and mature germ cells (oocytes and sperm). The first cells that bud out from the germ lineage are trophoectodermal cells that give rise to the placenta. Subsequently during gastrulation, EPSC are a source of pluripotent stem cells for all three germ layers (meso-, ecto-, and endoderm) and PGC. It is hypothesize that, at this stage, some EPSC could be deposited as Oct-4 pluripotent stem cells in peripheral tissues/organs (red circles). Similarly, some migrating PGC could go astray from their major migratory route to the genital ridges and become deposited as well. Furthermore, it is hypothesized that, similar to PGC, other EPSC deposited in the developing tissues undergo erasure of their somatic imprint (yellow arrows). This mechanism of erasure of methylation of somatic imprinted genes protects developing organism from the possibility of teratoma formation. However, it will affect some of the aspects of the pluripotentiality of these cells (e.g., potential of these cells to contribute to blastocyst development or teratoma formation after transplantation into immunodeficient mice). Reproduced with permission from Kucia et al. [40]

VSELs in Human Cord Blood

VSELs have been isolated from human cord blood by employing an isolation procedure that differs significantly from that usually used to obtain hematopoietic stem cells from cord blood. In the usual procedure for cord blood stem cell isolation, an important step is the removal of plasma from the collected sample, a process called volume depletion. However, this step was shown to remove a large percentage of the VSELs. Once a different isolation procedure was instituted, significant numbers of VSELs were identified in human cord blood. These cord blood-isolated VSELs are very small (5–7 microns) and highly enriched in a population of CXCR4+AC133+CD34+linCD45 cord blood mononuclear cells. These cells possess large nuclei that contain unorganized euchromatin and express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4 [45]. These studies indicate that VSELs, comparable to those from mice, are present in small but significant numbers in human cord blood. Further characterization of these cells with respect to pluripotency and other properties remains to be carried out

VSELs in Human Peripheral Blood and Bone Marrow

Significant quantities of VSELs can be obtained from the peripheral blood of humans following mobilization with G-CSF. Results show that after G-CSF mobilization, human peripheral blood contains a population of lin CD45 mononuclear cells that express CXCR4, CD34, and CD133. These CXCR4+ CD133+ CD34+ lin CD45 cells are highly enriched for mRNA for intra-nuclear pluripotent embryonic transcription factors such as Oct-4 and Nanog, and also express the cell surface marker SSEA-4, the early embryonic glycolipid antigen commonly used as a marker for undifferentiated pluripotent human embryonic stem cells. These human adult peripheral blood-derived VSELs, are larger than their counterparts identified in adult murine bone marrow, but are still very small (7–9 microns), and they also possess similarly large nuclei that contain embryonic-type unorganized euchromatin. Before G-CSF mobilization, very few VSELs were detectable in peripheral blood; following mobilization, there was a very significant increase in excess of 106 VSELs present in the apheresis product, representing much less than 0.0001% of total nucleated cells [46].

Very recently, Sovalat et al. [47] have confirmed the presence of a population of CD34+CD133+CXCR4+LinCD45 immature cells isolated from the human bone marrow and from apheresis product. Among this population of cells, they showed the presence of very small (2–5 microns) cells expressing Oct-4, Nanog and SSEA-4 at protein and mRNA levels. These investigators additionally showed that these cells express the receptor of SDF-1. As SDF-1 is secreted by the bone marrow microenvironment, the SDF-1/CXCR4 axis could play a critical role in accumulation of VSELs into the bone marrow. This study adds further support to the hypothesis that VSELs constitute a “mobile” pool of primitive/pluripotent stem cells that may be released from the bone marrow into the peripheral blood and have a pivotal role in tissue regeneration.

VSELs in Human Ovary and Testes

Two recent studies have established that VSELs can be isolated from human ovaries and testes, suggesting that, as in mice, VSELs are indeed present in niches throughout the organs of the human body [48, 49]. Bhartiya and colleagues have demonstrated that a population of small (5–10 microns), pluripotent stem cells are present in tissue derived from adult human testes. These cells, identified by in situ hybridization and immuno-localization, expressed Oct-4 and other makers of pluripotency, including Nanog and telomerase reverse transcriptase (TERT). It is reasonable to assume that these cells are VSELs and that they are responsible for the maintenance of normal testicular function. In addition, they are probably the same cells that have been described by He and others as capable of producing embryonic-like colonies from human adult testicular tissue [50, 51, 52, 53, 54]. The same workers have shown that two distinct populations of cells with similar properties to VSELs, and to those described above from human testis, can be isolated from the postmenopausal human ovarian surface epithelium as well as the adult rabbit, sheep, and monkey. The smaller 1–3 micron cells, characterized by the authors as VSELs, were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4–7 micron cells, with high nucleo-cytoplasmic ratio, cytoplasmic Oct-4 and minimal SSEA-4, were postulated to be daughter progenitor cells of the VSELs. The stem cell (VSEL) population underwent spontaneous differentiation into oocyte-like structures, parthenotes, embryoid body-like structures, cells with neuronal phenotype and embryonic stem cell-like colonies, whereas the larger cells transformed into mesenchymal phenotypes following 3 weeks in culture. This study not only establishes that pluripotent stem cells (VSELs) are present in the mammalian ovary but also establishes that, in agreement with earlier reports, the post menopausal human ovary, devoid of any follicles in the ovarian cortex, is a rich source of stem cells, which develop in vitro into oocyte-like structures [55, 56].

Regenerative Role of VSELs in Human Diseases and Conditions

The implication of a potential regenerative role of VSELs in human disease has been advanced by two recent studies: one conducted in patients who suffered a myocardial infarction [57] and one in patients who suffered a stroke [58].

Acute Myocardial Infarction

The first of these examined the mobilization of VSELs in patients who have suffered an acute myocardial infarction [57]. The authors of this study reported on 31 patients with acute myocardial infarction and compared the results obtained on these patients to 30 healthy subjects. In healthy subjects, the median number of circulating VSELs was very low. However, in patients with acute myocardial infarction, the number of VSELs increased in the peripheral blood in less than 12 h after the onset of symptoms, and remained elevated when measured after 24 h and 5 days. The mobilization of VSEL was significantly reduced in patients older than 50 years and in those with diabetes, as compared with younger and non-diabetic patients.

Circulating VSELs were small in size (6–8 microns) and compared well with the size of human VSELs reported by Medicetty et al. [46]. Characterization of these circulating VSELs also showed a comparable pattern of surface markers and intra-nuclear transcription factors to those found in G-CSF mobilized human adult peripheral blood. The number of circulating VSELs was shown to correlate with plasma SDF-1 levels, but there was no correlation with other cytokines, chemokines, growth factors, or inflammatory markers. The positive correlation between the plasma levels of SDF-1 and the number of circulating VSELs expressing the SDF-1 binding receptor CXCR-4 may have very important biological significance [59].

The negative correlation between the number of VSELs mobilized post infarction and diabetes is interesting since diabetes is associated with reduced mobilization, functional capacity, and survival of endothelial progenitor cells (EPCs), as well as promoting the senescence of cardiac stem cells [60, 61]. Of particular interest in the study by Wojakowski et al. [57] was the finding that the number of circulating VSELs present immediately post infarction had a positive correlation with the improvement in left ventricle ejection fraction (LVEF) at 1 year post infarct. This observation is consistent with previous studies that showed that the numbers of both CXCR4 and CD34 positive cells mobilized post infarct may be associated with improved recovery of LVEF, lower end-systolic volume, and decreased infarct size [62, 63, 64].


In the second study evaluating the potential regenerative role of VSELs in human disease, Paczkowska and her colleagues evaluated the number of circulating VSELs in the peripheral blood of 44 stroke patients and 22 age-matched controls [58]. After each patient’s stroke, peripheral blood samples were drawn during the first 24 h, on the third day, and on the seventh day, and compared with similarly obtained blood samples from the controls. The circulating VSELs were evaluated by real-time quantitative PCR, fluorescence-activated cell sorting analysis (FACS), and direct immunofluorescence staining. Serum concentrations of stromal derived factor-1 (SDF-1) were also analyzed by an enzyme-linked immunosorbent assay (ELISA). The stroke patients showed an increase in the total number of circulating stem cells, but more specifically, an increase in the number of circulating VSELs confirmed by their expression of the VSEL phenotype (CXCR4+linCD45 small cells), mRNA for Oct-4 and Nanog, and Oct-4 protein. As in the previous cardiac study, the increase in circulating VSELs was mirrored by an increased serum concentration of SDF-1. The number of circulating VSELs and the plasma levels of SDF-1 showed a positive correlation with the extensiveness of the stroke in all patients. This study suggests that stroke triggers the mobilization of VSELs and that the measurement of levels of circulating VSELs has a potential prognostic value in stroke patients.


The important observation that, in both humans and mice, VSELs decline in number, ability to mobilize, and other properties (e.g., sphere formation) has profound implications [45, 58]. For example, the number of circulating VSELs in steady state conditions in the peripheral blood of two-month-old mice was found to be five times higher compared to one-year-old mice (Fig. 8) [31, 65]. If the stored or mobilizable human VSELs are depleted with age in a way comparable to mice, then the concept of collecting and storing VSELs at an early age assumes great importance for future regenerative therapy. Since the magnitude of this effect appears to be under genetic control as shown by the finding that long-lived mice (C57BL/6) show much higher VSEL numbers in their bone marrow than short-lived mice (DBA/2J), it is of critical importance to identify genes that are responsible for tissue distribution/expansion of these cells as they could be involved in controlling the life span of mammals [65, 66, 67, 68].
Fig. 8

Multicolor flow cytometric analysis of VSEL content in fraction of nucleated cells isolated from bone marrow of 2-, 4-, 7-, 10-, 12-, 18-, 24- and 36-months old mice. Age-dependent decrease in the content of VSELs in murine bone marrow. VSEL content among bone marrow cells was analyzed as described in Fig. 2. Total nucleated bone marrow cells were stained for Sca-1, CD45, and lineage markers and subsequently analyzed by MoFlo. The graph shows the average content of VSELs according to the age of animals (Mean ± SEM). Reproduced with permission from Zuba-Surma et al. [65]

Ratajczak et al. have hypothesized that the pool of VSELs residing in adult tissues, including bone marrow, is regulated by circulating insulin-like growth factor-1 (IGF-1) levels. An increase in IGF-1 level (for example, resulting from a chronically high caloric uptake) would accelerate, in an Ins/IGF-dependent manner, an age-dependent reduction of the VSEL pool and, hence, their potential to rejuvenate tissues (for example, in bone marrow to supply long-term repopulating hematopoietic stem cells). A low circulating IGF-1 level (for example, as seen in Laron dwarf mutants or as a result of caloric restriction) would have an opposite and protective effect in maintaining the critical VSEL pool [69].

Animal studies are in progress to validate the capabilities of human VSELs to trans-differentiate into cardiomyocytes, and to facilitate cardiac angiogenesis [70, 71, 72, 73]. The role of VSELs in tissue regeneration for liver disease, retinopathies, and pulmonary conditions is under investigation [74, 75, 76, 77]. In addition, human VSELs are being studied in immune deficient animal models for bone regeneration, autoimmune diseases, wound healing, and nerve regeneration. The primary thrust of these experiments is the establishment of the pluripotency of human VSELs. Indeed, the recent paper by Yasumasa Kuroda et al. [78] not only provides further validation of the pluripotency of human adult stem cells but also raises again the possibility that the cells described by several authors as putatively pluripotent are either the same cells or closely related sub-populations. The cells described by the Kuroda group have so many analogous properties to human VSELs that a working hypothesis of equality is reasonable. The human “Muse” cells described by Kuroda not only can generate, from a single cell, cells with the characteristics of all three germ layers, but also express the pluripotency markers Oct3/4, Nanog, and Sox2. These cells are positive for SSEA3 and form cell clusters that seem comparable to embryoid bodies, a property not previously demonstrated for human VSELs. Like VSELs, these Muse cells have a low native proliferation activity and do not exhibit any tumorigenesis [69, 78]. Again, it should be emphasized that the demonstration of VSELs in human adult peripheral blood and in human bone marrow, opens the possibility of achieving all the positive benefits of the embryonic stem cells without the negative attributes such as tumorigenesis. Of even greater potential is the ability to obtain un-expanded pluripotent stem cells in quantities sufficient for therapies, and for autologous use, which is obviously impossible with embryonic cells. The lack of a reproducible capacity to expand VSELs to date might explain the limited recognition of these cells.

Induced Pluripotent Stem Cells

The production of pluripotent cells from non-pluripotent human adult somatic cells (initially fibroblasts) by scientists in Japan and the United States has caused great excitement. These cells have been called Induced Pluripotent Stem Cells (iPS) and are produced by forcing the cells to express certain genes. This reprogramming process is called retrodifferentiation, and is achieved by transfection (see below) of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts or mononuclear leucocytes. Transfection may be achieved through viral vectors, such as retroviruses and adenoviruses or, recently, non-viral vectors. Transfected genes include the master transcriptional regulators Oct-3/4 (Pouf51) and Sox2, although other genes and epigenetic factors can probably enhance the efficiency of induction. After 3–4 weeks, small numbers of transfected cells begin to adopt morphological and biochemical characteristics similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection.

iPSs are postulated to be functionally identical to natural pluripotent stem cells, such as embryonic stem cells, with respect to the expression of certain stem cell genes and proteins, chromatin methylation patterns and other epigenetic properties, doubling time, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability, but the full extent of their capabilities and limitations in comparison to natural pluripotent stem cells is still being assessed. iPSs were first produced in 2006 from mouse cells and in 2007 from human cells [79, 80, 81]. This has been cited as an important advancement in stem cell research, as it may allow researchers to obtain pluripotent stem cells, which are critical in research and potentially have autologous therapeutic uses, without the controversial use of embryos. However, there remain many problems to be solved before these cells could be used for therapeutic applications.

Methods to Produce iPSs

The first methods to produce iPSs required the use of retroviruses as the vectors to transfect cells to initiate the expression of the critical genes. This process would be unacceptable as a prelude to the production of cells for therapy since retroviruses are known cancer-causing agents [82]. The problem was then addressed through the use of a much safer adenovirus as the vector. However the efficiency of the process, already very low with the use of the retrovirus vector, is dramatically attenuated with an adenovirus vector, which certainly reduces tumorigenesis, but does not eliminate this problem, since the expression of endogenous oncogenes (proto-oncogenes) may potentially be triggered by the integration and harboring of exogenous sequences in the target cell genome.

Japanese researchers have discovered that the origin of mouse-derived iPS cells has a large effect on their tendency to trigger tumors, and that tumorigenic potential is related to the persistent presence of undifferentiated cells within the iPS-cell-derived cells [83]. The problem of tumorigenesis was elegantly addressed by a technique that can remove oncogenes after the induction of pluripotency, which, while removing or reducing the risk of tumor formation, greatly increases the complexity of the process [84]. In 2009 Sheng Ding and his colleagues at the Scripps Research Institute, La Jolla, California made the remarkable discovery that mouse iPS cells could be produced without any genetic alteration of the adult cell [85]. This advance was achieved by repeated treatment of the murine cells with four transcription factors, Oct4, Klf4, Sox2, and c-Myc, channeled into the cells via poly-arginine anchors to induce pluripotency. The acronym given for those iPS is piPS (protein-induced pluripotent stem cells). The extension of this work to human cells has not yet been reported. In addition, the production of iPS, whether by a viral vector or by protein induction and has been the subject of continuing concern on the basis that replacing oncogenes with the putative oncoproteins that are the product of the oncogenes may not eliminate the cancer risk on the same line of genetic-chemical flow of genetic information to phenotypic representation.

A method to greatly increase the efficiency of production of iPS has been proposed by Rossi and his colleagues [86]. They report methods to program multiple human cell types to pluripotency with efficiencies greatly exceeding those of piPS and viral techniques. In addition, this is achieved without modification of the genome to accomplish reprogramming of the cells. The reprogramming is accomplished by exposing the cells to repeated administration of synthetic messenger RNAs that incorporate modifications to circumvent the natural antiviral responses of the cells. They also show the same technology can be used to direct the differentiation of the reprogrammed cells, termed RiPSCs, into terminally differentiated myogenic cells. While the RiPSC technique increases the efficiency of stem cell production by approximately 100 fold as compared with viral techniques, it still only produces retro-differentiation in approximately 2% of cells treated.

Recently Lanza et al. investigated a range of cell types derived from eight human iPS cell lines and 25 embryonic stem cell lines [87]. Initial findings indicated that human iPS cells differentiated into blood vessel, blood precursor, and retinal cells with characteristics comparable to embryonic stem cells, but with significantly reduced efficiency. In addition the study found that cells derived from iPS cells had significantly higher rates of apoptosis than those derived from embryonic stem cells. Moreover, the hematopoietic and neural progenitors derived from iPS cells aged prematurely, lost their ability to divide, and showed a 1,000–5,000 fold lower proliferation rate than embryonic stem cells.

This reduced efficiency has been confirmed in studies published by Thomson and colleagues [88] who found that the two stem cell types behaved very similarly as they became neurons and glia, expressing the same genes at the same time, and both human embryonic stem cells and iPS-derived cells acted like normal brain cells in laboratory tests. But more than 90% of the human embryonic stem cells responded to the chemical recipe for making neural cells, whereas the iPS cells’ response was more variable—in some lines, only 15% of cells turned into neuronal cells, in another, 79% [88]. In a critical review of the epigenetic characteristics of iPS cells, Djuric and Ellis [89] examined the four stages of the reprogramming process, namely the starting differentiated cell, the intermediate cell, the partially reprogrammed iPS, and the fully reprogrammed iPS, with respect to seven criteria that must be satisfied to validate success (Fig. 9). Only partially and fully reprogrammed cells were capable of forming embryonic stem cell-like colonies that are commonly isolated during the clonal expansion of iPS cell lines.
Fig. 9

Dynamic epigenetic changes characterize the gradual reprogramming process. Four stages of reprogramming are illustrated as differentiated starting cell type (that is, murine embryonic fibroblasts), intermediate, partially reprogrammed induced pluripotent stem (iPS) cells, and fully reprogrammed iPS cells. Activation of pluripotency-associated loci results in iPS cells’ ability to stably self-renew and precisely control the embryonic stem cell (ESC)-like transcriptional profile. Proper histone methylation levels at the right genomic regions establish bivalent chromatin domains not necessarily involved in the induction to pluripotency but required for proper differentiation capacity of iPS cells. DNA methylation levels are reduced in female ESC and iPS cell lines at heterochromatic satellite repeat elements. The functional significance of DNA hypo-methylation is currently not understood but could be a reflection of the reactivated inactive-X chromosome. On the other hand, DNA methylation marks of imprinted genes remain protected from demethylation and are comparable to the levels in the starting cell type. Finally, it is not known whether extensive epigenetic changes accompanying the reprogramming process result in physical structural changes to the chromatin fibers themselves. Reproduced with permission from Djuric and Ellis [89]

The full reprogramming process is a rare event, with initial studies reporting that the reprogramming efficiencies in mice are 0.1% of the starting cells giving rise to visible colonies. This number is even smaller when considering only colonies that are fully reprogrammed with all the epigenetic characteristics of an embryonic stem cell-like state. Indeed, complete reprogramming is a rare event with most of the cells that initiate the reprogramming process failing to contribute to the germline of chimeric mice. The establishment of populations of partially reprogrammed iPS cells may have potential detrimental consequences with respect to tumorigenesis [89]. The very low efficiency of retrodifferentiation to generate iPS cells (less than 0.1% to 2%) has been stated to be surmountable by means of cell expansion whereby cells are grown in a culture medium to increase their numbers. However, this procedure is only effective for a very small number of passages (cell divisions), probably less than five, after which the cells show decreased potency. Moreover, the expandability of cells that have undergone retrodifferentiation has not been studied.

Clinical Implications of Human iPS Cells

The generation of human iPS cells by direct reprogramming of human somatic cells and the generation of disease-specific iPS cells from patients with a number of pathologies have very important implications for investigating the mechanisms of cell reprogramming and for probing aspects of human disorders. For example, the reader is referred to the study by Moretti et al., on the use of patient-specific iPS derived from a family with long-QT syndrome [90, 91]. However, the production of these cells from human fibroblasts or other cells is extremely demanding from a scientific and technical viewpoint. That, in turn, requires a very high quality production facility. The combination of these factors, together with the extreme inefficiency of all current iPS methods, means that it would take a long time to achieve an acceptable cell-dose, making the use of iPSs for therapy not only cost-prohibitive but also functionally impractical. In addition, there can be little doubt that regulatory agencies will be inevitably and appropriately demanding in their requirement for extensive safety data and rigorous risk evaluations before consideration of iPSs for human use could be initiated (Fig. 9). It is also of interest that recent work by Hochedlinger and others would suggest that there is commonality between the restricted abilities of iPS and the non-teratoma forming attributes of VSELs [92]. The work points to the possible role of the epigenetic control of the hypo- and hyper methylation of paternal and maternal imprinted genes as the basis for the erratic functioning of iPS as compared to embryonic stem cells.


It is evident that, for the foreseeable future, therapy development and performance will be based on, and dependent upon, the availability of sufficient quantities of autologous cells with high levels of plasticity. This can only be achieved at this time through the use of non-manipulated cells collected by minimally invasive procedures, such as apheresis, from the peripheral blood following mobilization or bone marrow aspiration. Of the available cell choices, current evidence is rapidly establishing the central role of the VSEL as the optimal candidate cell for therapy development. Recent studies have confirmed that significant quantities of VSELs can be obtained from the peripheral blood of humans following mobilization with G-CSF. Thus, the ability to obtain autologous stem cells at more than adequate cell doses points to the potential central role of VSELs as the regenerative cell of choice. This is especially true since VSELs share many of the attributes of iPS cells with none of the negative characteristics or economic and technical limitations. While definitive confirmation that VSELs have multilineage in vivo regenerative properties, is still lacking several groups have shown that VSELs indeed can contribute to tissue repair that could be explained by combinations of several factors, including transdifferentiation, protection and the recruitment of endogenous stem cells. With the possible exception of the cell clusters, which seem comparable to embryoid bodies described by Kuroda et al.(78), embryoid body formation in human VSELs has yet to be established. Possible explanations include the age of the donor, since VSELs from older mice lose the ability to form embryoid bodies, or in finding the right combination of growth factors and culture conditions. VSELs appear to have many of the attributes of embryonic stem cells with none of the moral, religious, political, or ethical restrictions.



This review was supported in part by grant R43 AR056893-01A1 from NIAMS. The authors gratefully acknowledge the review of the manuscript by Dr. Mariusz Z. Ratajczak, Henry S. and Stella M. Hoenig Professor, Department of Medicine, University of Louisville School of Medicine, Louisville, Kentucky. Editorial assistance was provided by Jan S. Redfern, PhD, Redfern Strategic Medical Communications, Inc., Goshen, NY.

Conflict of Interest Statement

The authors are employees of, and own stock in, NeoStem, Inc. D.O.R. is Director of Stem Cell Science. A.G.H. is Vice President of Regenerative Medicine, Drug Development & Regulatory Affairs


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.NeoStem, Inc.New YorkUSA

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