Life in plastic is fantastic

The claim that cells with transgermal potential exist in postnatal tissues raises major biological and experimental questions.

In this issue of Blood, Beltrami and colleagues report that nonhematopoietic cells expressing markers of mesenchymal stem cells isolated from human adult bone marrow, liver, and heart can be induced to proliferate extensively, cloned after culture, and pushed to express features that are normally associated, in vivo, with derivatives of the 3 germ layers. The properties and culturing of these cells recall MAPCs (multipotent adult progenitor cells), described earlier in human and rodent bone marrow.¹  To some, the data might illustrate the effects of culture conditions. To others, the data might suggest that cells with properties unique to pregastrulation embryonic cells and embryonic stem (ES) cells can be found in adult organs and made available in large quantities, dispelling the need to even consider human ES-cell research. Beltrami and colleagues remain cognizant that the properties of their cells can reflect a culture artifact. Nonetheless, they choose to name their cells multipotent adult stem cells (MASCs), and suggest that regardless of whether their MASCs are fact or artifact, they could be useful for regenerative medicine.

Nonhematopoietic stem cells, unlike hematopoietic stem cells (HSCs), can give rise to cultured cell strains. This circumstance sometimes confounds in vitro phenomena and defining properties of stem cells. The latter (self-renewal, organogenic capacity) are only relevant to “stemness” and physiology when assessed in vivo.

The ability of a cell to self-renew, while at the same time generating an expanding, differentiating progeny, is proven through in vivo transplantation and recovery from the recipient's tissue of cells identical to those transplanted. Sustained expansion or clonogenicity in culture do not surrogate self-renewal. An impressive number of population doublings in vitro (40 for MASCs) does not make a cell a stem cell. The HSC, the only stem cell for which self-renewal is generally considered proven, does not grow in culture. Clonogenicity is a property of stem cells, but is neither unique nor defining. Only a small proportion of freshly isolated mesenchymal cells display the ability to initiate clonal growth at low density (∼1 cell/cm²), which most cells cannot do. Mesenchymal cells, however, can acquire clonogenicity through proliferation in culture. Nonclonal lines of MASCs do not grow when seeded at less than 500 cells/cm² (higher than clonal density). After culture with mitogens, however, approximately 17% of cells in the same lines generate clones when seeded at approximately 1 cell/cm². From the expression of divergent differentiation markers in the clones, the authors infer single-cell multipotency. However, detection of multipotency is contingent on clonal growth, which is induced by culture, like differentiation potency itself can be. Although the authors could not grow multipotent cells from blood, blood does not compare with bone marrow or solid-phase organs in content of adherent mesenchymal cells and cannot provide a control for the effects of culture.

Although the pangermal potency of ES cells is proven in vivo, Beltrami and colleagues deduce a transgermal differentiation potential of MASCs from in vitro assays. Validation of an in vitro differentiation assay should come from an independent in vivo assay, but this is rarely the case. Bone marrow stroma can generate histology-proven bone in vivo, with no induction in culture. However, expression of osteoblastic markers can be induced in culture in a variety of cells that remain unable to make bone in vivo. Likewise, OCT-4 pseudogenes expressed in postembryonic cultured cells do not signify an embryonic potency. All epigenetic states are reversible. Therefore, turning a fibroblast into a hepatocyte-like cell through in vitro conditions is conceivable and possibly feasible, but does not make fibroblasts progenitors of hepatocytes, nor true hepatocytes the progeny of fibroblasts. A fibroblast pushed to express hepatocyte features (one or several) in vitro might not survive if transplanted in vivo, or might lose the properties acquired in vitro. In the absence of proof of in vivo differentiation, no claim of potency rests on proper evidence.

Given the lack of evidence for self-renewal and multipotency, and in the absence of perspective on the in vivo identity, origin, and physiological role of any cell, assuming a stem-cell nature may not be prudent. Contemplating a potential clinical application (which is not only in vivo, but pro vivo) for cells never tested, and possibly not existing in vivo may be less than prudent. The path to the definition of the identity and properties of HSCs provides an important in vivo experimental paradigm. To make progress, research on nonhematopoietic stem cells should adapt the paradigm to the logic of the systems under study, but treasure its fundamental lessons.