Concise Review: Culture Mediated Changes in Fate and/or Potency of Stem Cells§


  • Disclosure of potential conflicts of interest is found at the end of this article.

  • Author contributions: V.D.R., K.V, and C.M.V.: manuscript writing, final approval of manuscript. V.D.R. and K.V. contributed equally to this article.

  • §

    First published online in STEM CELLSEXPRESS February 8, 2011.


Although Gurdon demonstrated already in 1958 that the nucleus of intestinal epithelial cells could be reprogrammed to give rise to adult frogs, the field of cellular reprogramming has only recently come of age with the description by Takahashi and Yamanaka in 2006, which defined transcription factors can reprogram fibroblasts to an embryonic stem cell-like fate. With the mounting interest in the use of human pluripotent stem cells and culture-expanded somatic stem/progenitor cells, such as mesenchymal stem cells, increasing attention has been given to the effect of changes in the in vitro microenvironment on the fate of stem cells. These studies have demonstrated that changes in culture conditions may change the potency of pluripotent stem cells or reprogram adult stem/progenitor cells to endow them with a broader differentiation potential. The mechanisms underlying these fate and potency changes by ex vivo culture should be further investigated and considered when designing clinical therapies with stem/progenitor cells. STEM CELLS 2011;29:583–589


Development is believed to occur by sequential discrete changes in stem/progenitor cells associated with loss of lineage potential but increase in the degree of specification toward ultimately a single differentiated cell. Over the years, it has become clear that these discrete changes in cell fate are orchestrated by epigenetic mechanisms leading to silencing of pluripotency genes and activation of developmental genes [1].

In 1957, Waddington [2] suggested that differentiation can be compared with a ball rolling through different one-way branched valleys between hills, yielding ultimately an irreversible cellular fate. However, evidence that the differentiation process is reversible already existed in 1958, when Gurdon et al. [3] demonstrated that transfer of intestinal epithelium cell nuclei from albino tadpoles into unfertilized wild-type eggs resulted in the development of adult albino frogs. The same technique, termed somatic cell nuclear transfer (SCNT), was used to generate “Dolly” the sheep [4] and many other species since. The nature of the factor(s) present in unfertilized eggs capable of resetting the epigenetic landscape from a terminally differentiated cell to a fully totipotent state remains elusive. Apart from oocytes, embryonic germ (EG) cells and embryonic stem cells (ESC) also contain factors that can reprogram somatic cell nuclei, as fusion of these cells with somatic cells creates tetraploid hybrids wherein silencing and activation of genes on somatic chromosomes occurs [5]. Although transcriptional differences exist between ESC and ESC/EG cell-somatic cell hybrids [6], the latter can generate embryoid bodies (EBs) and teratomas and contribute to chimeras when injected into the blastocyst [7, 8]. The activation of the pluripotency-associated transcription factor (TF) Oct4 can also be achieved through the introduction of cell-free extracts from ESC and embryonic carcinoma (EC) cells, although full reprogramming to pluripotency has not been shown [9].

The field of cellular reprogramming took a very large step forward in 2006, when Takahashi and Yamanaka [10] demonstrated that retroviral transfer of only four TFs (Oct4, Sox2, Klf4, and c-Myc) could convert murine somatic cells into ESC-like cells, termed induced pluripotent stem cells (iPSCs). Since this first report, many cell types of different maturity stages and species have been reprogrammed to a pluripotent stage by a similar combination of factors, demonstrating the universal nature of the process [11]. During the last 4 years, the role of and the need for the different TFs originally used have been further elucidated and techniques have been developed to generate iPSC without integrating vectors [12]. Although it is clear that the reprogramming factors must enable cells to push backward in the so-called valleys described by Waddington [2], thereby removing the epigenetic hurdles that stabilize cells in their differentiated status, we still have a long way to go before we comprehend the epigenetic changes required for full reprogramming. Nevertheless, several small molecules, including epigenetic modifiers and signaling pathway inhibitors, have been used successfully to enhance reprogramming or replace TFs to the point where all original TFs, except Oct4, can be replaced by a cocktail of small molecules [12, 13].

Although complete dedifferentiation from a somatic to a pluripotent state still requires the introduction of one or more TFs, reports of more modest dedifferentiation events demonstrate that reprogramming, to some extent, can be achieved through manipulation of the culture microenvironment alone.


That the culture microenvironment may affect the epigenetic landscape and transcriptome of ESC and iPSC has recently gained significant attention. Establishment of ESC from the inner cell mass (ICM) requires some form of epigenetic reprogramming such that the genes that confer a growth advantage might be upregulated and the genes nonessential for in vitro culture might be silenced [14]. Upon long-term culture of ESC, epigenetic changes occur that are stably inherited by the cell line. For instance, culture on Matrigel rather than feeders, different passaging methods, use of serum substitutes, or culture in hypoxic conditions appear to affect the transcriptome and/or epigenome of ESC [14, 15]. Further evidence that in vitro culture can influence the fate of stem cells comes from recent studies demonstrating that specific culture conditions, with or without epigenetic or signaling pathway modifiers, can reversibly convert epiblast stem cells (EpiSC) or primitive ectoderm (PrE)-like cells to an ICM-like mouse ESC (mESC) fate.

Hayashi et al. [16] demonstrated that mESC cultures are heterogeneous and that ICM-like mESC can adopt in a reversible manner an epiblast-like fate during culture. Although conversion of an ICM-like to an epiblast-like fate merely reflects the progression of cells along an inherent developmental program, conversion of an epiblast-like to an ICM-like fate reflects dedifferentiation to a developmentally more immature fate. This interconversion is influenced by environmental cues as maintenance of Stella-positive ICM-like cells without mouse embryonic fibroblasts (MEFs) leads to the commitment to a Stella-negative epiblast-like state, which is reversed when cells are plated again onto feeders. Consistent with the fact that gain and loss of Stella gene expression is accompanied by changes in histone acetylation but not DNA methylation of the Stella locus, addition of trichostatin A, a histone deacetylase inhibitor, reversibly converts the cells to a Stella-positive ICM-like state. As another example, Toyooka et al. [17] demonstrated that mESC cultures contain Rex1-positive ICM-like cells and Rex1-negative early PrE-like cells. Similar to what was shown for the heterogeneous mESC cultures described by Hayashi et al., clonal Rex1-negative cells could reversibly be converted into Rex1-positive cells, in part influenced by the culture conditions used.

Similar evidence exists that EpiSC isolated from the postimplantation epiblast of E5.5 to E7.5 mouse embryos can be reverted to the ICM-like ground state. In an initial study, short-term forced expression of Klf4 lead to the creation of true pluripotent stem cells that generated germline-competent chimeras, which was associated with loss of X chromosome silencing [18]. In a second series of studies, similar dedifferentiation from an EpiSC to an ICM-like state was accomplished by supplementing standard mESC medium, containing leukemia inhibitory factor (LIF), with small molecules targeting epigenetic modifiers and/or signaling pathways differentially used by the two pluripotent cell types [19, 20]. Moreover, Bao et al. [21] demonstrated that EpiSC derived from E5.5 to E7.5 embryos could spontaneously revert to an ICM-like ground state when cultured for 14–35 days on MEFs supplemented with LIF but without any other factors. The reverted ESC could generate germline-competent chimeras and progressively lost the histone 3 lysine 27 trimethylation (H3K27me3) mark on the inactive X chromosome, which was associated with loss of X-chromosome silencing.

Taken together, these reports clearly demonstrate that manipulation of the culture microenvironment can cause EpiSC to dedifferentiate to a more primitive ground state. This notion also holds true for stem cells harvested beyond the epiblast stage, which will be discussed below.


Adult stem cells have a more restricted differentiation potential than ESC/iPSC. However, over the last decade several studies have reported unexpected greater potency of cultured postnatal stem cells. Initially, it was believed that these cells were not created by reprogramming events, as no major manipulations were required to obtain cells with potency different than the population they were isolated from. Therefore, it was hypothesized that these more potent stem cells were selected in culture from among the tissue specific stem/progenitor cells present in a given tissue. However, the inability to isolate cells with such higher potency directly from the somatic tissue they originate from, together with the recent evidence that reprogramming can be achieved by defined factors, has changed our perspective. It appears more and more clear that the culture microenvironment, wherein postnatal stem cells are maintained, can affect cell fate and causes dedifferentiation in some cases to cells with ESC-like potency.

Can In Vitro Culture Convert Germline Stem/Progenitor Cells to Cells with Pluripotent Characteristics?

In 2004, Kanatsu-Shinohara et al. [22] reported for the first time that when germline stem cells (GSC also referred to as spermatogonial stem cells [SSC]) from neonatal mouse testis were cultured in the presence of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), and LIF, occasional colonies with ESC morphology appeared within 4–7 weeks after initiation of the culture. These colonies could subsequently be maintained with 15% serum and LIF, that is, classical mESC culture conditions, and were termed multipotent germ stem (mGS) cells. The efficiency of generating mGS cells was significantly greater when GSC from p53 knockout mice were used, consistent with the observation that reprogramming adult cells to iPSC is more efficient when p53 knockout cells are used, and inhibited by activated p53 [23, 24]. The mGS cells were SSEA1+ and expressed Sox2 and Nanog besides Oct4 at levels near those in mESC. Moreover, mGS cells had lost spermatogonial potential and could now form teratomas and contribute to chimeras with germline transmission. When compared with GSC, mGS cells had undergone partial erasure of the typical androgenetic imprinting pattern. Although mGS cells are very much alike to mESC, live offspring from tetraploid complementation experiments could not be derived.

In 2006, Guan et al. [25] reported that Oct4, Nanog, and Sox2 positive cells could be generated from adult mouse testis as well. When Stra8+ SSC were cultured under standard ESC conditions, tightly packed ESC-like colonies appeared (multipotent adult GSC [maGSC]). Like mGS cells and ESCs, maGSC form EBs and teratomas but chimeric contribution was not tested. Seandel et al. [26] reported that culture of adult testis-derived GPR125+ spermatogonial progenitors on mouse testicular stroma cells for 2–3 months generated distinct colonies of multipotent adult spermatogonial-derived stem cells (MASCs), which expressed Sox2 and Nanog besides Oct4 and could be maintained in culture under routine mESC conditions. The transcriptome of MASCs differed from that of the original spermatogonial progenitor cells and from that of mESC. Nevertheless, MASCs generated derivatives of the three germ layers in vitro and teratomas in vivo, and contributed partially to chimeric embryos. However, germline transmission was not seen and, therefore, none of the adult testis-derived cell types (maGSC and MASCs) can be considered fully pluripotent. Because these studies were done using (enriched) populations of testis-derived cells, the possibility existed that the outgrowth of cells with ESC-like properties reflected mere selection of rare pluripotent stem cells retained in the testis postnatally. In 2008, Kanatsu-Shinohara et al. [27] demonstrated that a single testis-derived cell could generate both GSC and mGS cells, providing evidence that a cell committed to a single lineage can revert to a pluripotent state. However, the efficiency of such spontaneous dedifferentiation was low. In 2009, Ko et al. [28] reported that clonally established, unipotent GSC from adult testis, after 3–4 weeks in a specific culture microenvironment, gave rise to ESC-like colonies that could subsequently be maintained under ESC conditions. Pluripotency of these germline-derived pluripotent stem (gPS) cells was confirmed by gene expression profile, teratoma formation, germline contribution in chimeras, and germline transmission to the next generation. However, tetraploid complementation experiments did not result in born pups, probably due to persistent androgenetic imprinting status of gPS cells.

In 2008, Conrad et al. [29] described that cells derived from human adult testis could also be converted to cells with ESC-like properties. Single cells from adult testis were cultured with knockout medium and GDNF for 4 days, followed by CD49f preselection and further matrix selection with laminin. Human adult GSC (haGSC) colonies appeared after 21–28 days of subsequent culture in basic medium with LIF. Like human ESC (hESC), haGSC expressed SSEA4, TRA 1–60, TRA 1–81, OCT4, NANOG, and SOX2 proteins and formed EBs and teratomas. However, transcriptome studies demonstrated that expression of OCT4, NANOG, and SOX2 was significantly lower in haGSC than in hESC, consistent with the notion that the promoter regions of OCT4 and NANOG were only partially demethylated in haGSC. As full demethylation of these promoters is an important criterion of complete pluripotency [30, 31], this suggests that haGSC may only have acquired a partially reprogrammed state.

In summary, all these studies strongly suggest that germline stem/progenitor cells from mouse testis can be converted into ESC-like cells without the introduction of exogenous reprogramming factors, but by (long-term) culture under specific in vitro conditions. However, transcriptional differences exist with ESCs, androgenetic epigenetic marks are not fully erased, and tetraploid complementation has not been achieved. The dedifferentiation of human GSC to cells with ESC features is currently less robust and awaits further confirmation [32]. Whether the conversion is induced by the culture medium itself or is a stochastic epigenetic event that is then captured and further selected by the culture medium remains to be determined. As Ko et al. [28] found that conversion of testis-derived cells (0.01%) was only possible when cells were plated at specific densities, one might speculate that the culture conditions themselves play a role in this dedifferentiation process. It is worth noting that GSC are the only postnatal cells that continue to express high levels of Oct4, the only TF that cannot yet be replaced by other TFs or small molecules for the generation of iPSC [13]. Moreover, clinical teratomas, which contain cells from the three germ layers, occur almost exclusively in the gonads, and during development, EG cells, derived from the genital ridge that ultimately generate GSC, have a differentiation potential similar to that of ESC [33]. Therefore, GSC may be more permissive for conversion into pluripotent cells than any other cell type.

Because in contrast to germline stem/progenitor cells, somatic stem cells do not express Oct4 and their ability to self-renew and differentiate to mature cells does not depend on Oct4 [34], the question arises whether adult stem cells can also be converted to cells with ESC-like properties without introduction of defined factors.

Can the Culture Conditions Endow MSC with Greater Potency?

In this section, studies that have suggested that mesenchymal stem cells (MSC) can acquire a greater potency under specific culture conditions will be critically examined. MSC were first described by Friedenstein et al. [35], who demonstrated that besides hematopoietic stem/progenitor cells, bone marrow (BM) also contained cells that had the appearance of fibroblasts when cultured adherent to plastic. These cells, initially named fibroblast colony forming cells, differentiate into fibroblasts, adipocytes, chondrocytes, and osteocytes and were later renamed as MSC by Caplan and coworkers [35]. Genome-wide transcriptome and promoter methylation studies have shown that the expressed gene profile changes on culturing MSC in vitro with increased expression of, among others, cytoskeleton proteins and downregulation of cell cycle inhibitory genes [36], which is accompanied by progressive epigenetic changes [37]. There is also evidence that relatively minor changes to the culture conditions used to expand MSC, including culture at different oxygen tensions, use of fetal calf serum versus autologous serum, use of platelet lysate, or addition of growth factors (e.g., FGF2 or platelet-derived growth factor [PDGF]), affect MSC proliferation ability as well as the rate and degree of differentiation to the osteogenic, adipogenic, and chondrogenic lineages [36, 38–41]. These functional changes are associated with differences in expressed gene profile and epigenomic state of the cells [36, 38–41].

Other studies have suggested that cultivation of MSC from BM, cord blood, amniotic fluid, liver, and heart, among others, yields cells with broader differentiation potential than classically ascribed to MSC. In 2002, we described multipotent adult progenitor cells (MAPC) derived from postnatal rodent BM [42]. In contrast to MSC, rodent MAPC, cultured at low density and in the presence of LIF, EGF, and PDGF, differentiated in vitro to cells of the three germ layers and one of the murine lines contributed to chimeric mice, although in general contribution was very low and no germline transmission was seen. Recent isolations of rodent MAPCs, which express the ESC-associated TF Oct4 (but not Nanog and Sox2) and the primitive endoderm specific genes Gata6, Gata4, Sox7, and Sox17, demonstrate that they can be detected after 2–4 months of in vitro culture [43, 44]. As such, rodent MAPC resemble extraembryonic endoderm cells [45] and extraembryonic endoderm precursor cells [46]. In the initial report, we speculated that MAPC might be embryonic remnants or the result of dedifferentiation of a BM derived cell to a more potent cell. Studies are currently ongoing to determine which of these two hypotheses is correct (manuscript in preparation).

That cells with MAPC properties may be (in part) generated in vitro comes from a study by Anjos-Afonso and Bonnet, who demonstrated that when SSEA1+ cells isolated from fresh Lin/CD45/CD31neg BM cells were cultured under MAPC conditions, expression of Oct4, Nanog, and Rex1, present at low levels in the freshly isolated cells, increased significantly. Moreover, the cultured cell population displayed, like MAPC, differentiation potential toward mesoderm, endoderm, and ectoderm in vitro, as well as several mesodermal lineages, including hematopoietic cells, in vivo [47]. Noteworthy, when the SSEA1+ BM cells were cultured under MSC conditions, the levels of Oct4 and Nanog expression could not be maintained. Also, when the SSEA1 cell fraction was cultured under MAPC conditions, no expression of these pluripotency-related TFs was seen, suggesting that perhaps only some cells within BM cell cultures can be reprogrammed to a more immature state.

Since the original description of rodent MAPC, other groups and we have published that cells with broader differentiation potential can also be culture-isolated from human tissues, including BM (MAPC, marrow isolated adult multilineage inducible [MIAMI] cells and BM-derived multipotent stem cells [BMSC]), from heart, liver, and BM (multipotent adult stem cells [MASC]), from amniotic fluid (amniotic fluid-derived stem [AFS] cells), from peripheral blood (multipotent progenitor cells [MPC]), and from cord blood (unrestricted somatic stem cells [USSC]) [48–52]. The cell surface phenotype, TF expression profile and differentiation potential of these cells is summarized in Table 1.

Among the different cell populations, some (MAPC, MASC, MIAMI cells, AFS cells, and MPC) but not all (BMSC and USSC) were reported to express one or more of the pluripotency-associated TFs. However, the data should be interpreted with caution, as multiple pseudogenes and/or isoforms exist for human OCT4 and NANOG, which do not confer pluripotency, but are detected by reverse transcriptase polymerase chain reaction and by most antibodies [53]. Therefore, expression levels should always be compared with a positive (hESC or human EC [hEC] cells) and negative (mature somatic cells) control, which was not performed in any of the studies above.

A second word of caution is needed when assessing the degree of differentiation obtained from these different cell populations. Demonstration of differentiation from stem cells requires not only that lineage specific transcripts and proteins are expressed following differentiation schemes in vitro but also that the cells acquire functional characteristics of the presumed differentiated progeny. In addition, when assessing differentiation in vivo, functional contribution to a given tissue is required. As detailed in Table 1, for many of the cell populations, differentiation into cells with functional endothelial, hepatocyte, and neuron-like features was demonstrated in vitro. Although many studies reported engraftment or differentiation in vivo, contribution to functional cell types different from the mesenchymal lineages such as endothelial or hematopoietic cells was only demonstrated for rodent and human MAPC.

Table 1. Overview of the different mesenchymal stem cell populations that display greater potency
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Taken together, all of the above described MSC-like populations are endowed with a broader differentiation capacity than “classical” MSC but none of them can be considered pluripotent as they do not form EBs, teratomas, or germline-competent chimeras. The functional differences with MSC (or more mature cell types) and/or embryonic cell types (ESC/EC cells) are also reflected in transcriptome and epigenome studies carried out with MAPC, USSC, MASC, and MPC [43, 48, 66, 69, 70]. For example, a transcriptome and epigenetic analysis of human MAPC (hMAPC), human MSC (hMSC), and hEC cells have demonstrated significant differences among the three cell populations [69]. In hEC cells, strong epigenetic repression of differentiation genes was seen, which was associated with both DNA methylation and histone modifications; whereas, in hMSC, differentiation genes showed active epigenetic profiles. In hMAPC, which have broader differentiation potential than hMSC but significantly less than embryonic cells, expression of differentiation genes was partially suppressed, associated with only repressive histone modification marks but not DNA methylation.

Although there is currently no formal proof that the greater potency of these cell populations is the result of reprogramming in vitro, there is also no evidence that cells with these characteristics exist in vivo. As indicated above, the study by Anjos-Afonso and Bonnet suggests that at least some of the potency may be acquired in vitro. Culture conditions for all these cell populations are relatively similar, although differences exist in cytokines, serum, and oxygen levels used. Whether this variation is responsible for the differences between MAPC, MIAMI cells, BMSC, MASC, AFS cells, MPC, and USSC populations is also unknown. If ex vivo culture leads to dedifferentiation from an MSC fate to cells with a more immature cell fate, it also remains to be determined that the conversion is induced by the culture medium itself, rather than via stochastic epigenetic events that are sustained by the culture medium.


Although it was believed that development occurs through sequential, irreversible fate changes in stem/progenitor cells resulting in loss of lineage potential and gain in specification toward ultimately mature cells, it has become clear that dedifferentiation can occur. Numerous studies demonstrated that dedifferentiation can be achieved by SCNT, fusion with ESC/EG cells, or defined TFs (Fig. 1). In addition, there is mounting evidence that changes in the ex vivo microenvironment wherein stem/progenitor cells are maintained can induce dedifferentiation of stem/progenitor cells to a fate that precedes the developmental stage of these stem/progenitor cells (Fig. 1). The precise epigenetic and consequent transcriptome changes underlying such fate changes—whether via SCNT, fusion, defined TFs, or culture conditions—remain to be fully clarified.

Figure 1.

Schematic overview of different reprogramming methods (A): Dedifferentiation via somatic cell nuclear transfer, fusion or defined factors toward a totipotent or pluripotent state. (B): Alterations in the potency of stem/progenitor cells as a result of the culture microenvironment. (i): Interconversion between epiblast stem cells and embryonic stem cells. (ii): Dedifferentiation of germline stem/progenitor cells toward a pluripotent state. (iii): Cells of mesodermal origin (e.g., mesenchymal stem cells) may gain a greater potency allowing differentiation to cells of the three germ layers. Abbreviations: EpiSC-ESC, epiblast stem cells-embryonic stem cells; ICM, inner cell mass; iPSC, induced pluripotent stem cells; SCNT, somatic cell nuclear transfer.


This work was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen) (to V.D.R. and K.V.); the Centre of Excellence and Stem Cell Center Program Funding K.U.Leuven and Odysseus Funding from the Fonds voor Wetenschappelijk Onderzoek in Vlaanderen (FWO Vlaanderen) (to C.M.V.).

Disclosure of Potential Conflicts of Interest

C.M.V. is a consultant for ReGenesys and for Athersys, Inc. and receives funding from Athersys, Inc. V.D.R. and K.V. indicate no potential conflicts of interest.