Although hematopoietic stem cells (HSCs) represent a minute percentage of the bone marrow compartment, they are known to reconstitute all blood-forming lineages. HSCs can also be found in the cord blood, which offers an easily bankable source that has already been proven in the clinical arena. More recently, a number of studies have shown that the bone marrow and cord blood host multipotent cells with the ability to differentiate into many different tissues. Certainly, MSCs are one such multipotent cell type, and perhaps the main component of the subpopulations selected for attachment and growth on plastic. In this context, it has been described that cord blood-derived MSCs subjected to a chemical differentiation protocol including high glucose, retinoic acid, nicotinamide, epidermal growth factor, and exendin-4 leads to the generation of ICCs expressing insulin, glucagon, GLUT-2, Pdx1, Pax4, and Ngn3. These cells, however, fail to regulate insulin secretion in response to glucose challenge in vitro. Similar results were recently presented by Chao et al., whose treatment of MSCs derived from the Wharton’s jelly (a stem cell-rich mucopolysaccharide layer within the cord blood) with neuron-conditioned medium resulted in the formation of ICCs positive for glucose-regulated human C-peptide secretion. When implanted in nonimmunosuppressed diabetic rats, these cells engrafted successfully and ameliorated hyperglycemia.
As for the non-MSC compartment, HSCs are presently being used for the treatment of diabetes in clinical trials for diabetes, but from a different angle: the reeducation of the immune system. In clinical trials conducted on 15 newly diagnosed type I diabetic patients, HSCs were mobilized, collected from peripheral blood, and cryopreserved. Patients were then subjected to a very aggressive immunosuppressive regime prior to the reinfusion of their own stem cells, which were assumed to be at a stage prior to the onset of the disease. As a result of this treatment, 14 out of 15 patients became insulin-free for extended periods of time. Others are already transplanting hematopoietic cells directly into the pancreas of diabetic patients, but the claimed beneficial effects of this treatment on the overall glycemic control still need to be independently validated– and a mechanistic explanation investigated. As was the case with MSCs, since the hematopoietic compartment is derived from the mesoderm, there are doubts that HSCs may become true endodermal or ectodermal derivatives. To this date, there is no conclusive evidence in one way or another that transplanted HSCs that migrate to target tissues help in their regeneration by differentiation/ transdifferentiation, cell fusion, or simply by supporting endogenous regeneration through revascularization and/or as feeders.
The latest cell type to join our arsenal of adult stem cells is the amniotic fluid stem (AFS) cell. These cells are naturally shed by the embryo to the surrounding amniotic fluid, and can be easily cultured in vitro. In a recent breakthrough report, De Coppi et al. described a novel subpopulation of amniotic fluid cells with a multilineage differentiation potential spanning the three embryonal layers. These cells can be expanded almost indefinitely without loss of pluripotency, and they coexpress markers of both human embryonic stem cells (Oct3/4, SSEA-4, telomerase, and others) and MSCs (CD105, CD73, and CD90). Unlike huES cells, however, AFSs did not form teratomas in immunocompromised recipients. Their proven ability to generate endodermal derivatives bodes well for the field of pancreatic regeneration, although current efforts at differentiating these cells into pancreatic endocrine cell lineages have not been reported yet.
Transiently Immortalized Beta Cells
A potential alternative to the use of adult stem cells for directed differentiation into beta cells would be to define the conditions to expand fully differentiated beta cells. we have already reviewed the literature on beta cell regeneration in vivo. However, in vitro “re-differentiation” of beta cells after de-differentiation and expansion phases was a less than convincing strategy, due to the fact that the end product could not stand comparison with the real beta cell. A different approach to the same end was presented in 2005 by Narushima et al., who transfected primary human beta cells with loxP-flanked sequences for the simian virus 40 large T antigen (SV40T) and the human telomerase reverse transcriptase (hTERT). These cells could be expanded without senescence for more than 50 passages in vitro. During the expansion phase, expression of insulin and other critical beta cell genes was downregulated. However, upon Cre-mediated “reversion,” the two immortalizing genes were removed and the cell adopted a quiescent, functional beta cell phenotype again. Reverted cells were able to permanently correct hyperglycemia in streptozotocin-treated SCID mice without any further replication in vivo . A complementary technology that could help the clinical translation of these findings is that of transducible TAT-Cre whose use in this context would preclude the use of viral vehicles to deliver the recombinase.
Transient immortalization of beta cells, as described by Narushima et al. A double round of retroviral transfection allows for the selection of cells that express both the hTERT and the SV40 large T antigen genes, which confer immortality. An EGFP marker can be used to sort these cells. This can be reverted by introducing the Cre recombinase, which will excise out the immortalizing genes in a process that will bring together a constitutive promoter and a neomycinselectable marker. These “reverted” cells can be selected using G418 and by sorting for EGFPnegative signal.
Tags: cord blood host multipotent cells, Cord blood stem cells, epidermal growth factor, generation of ICCs expressing insulin, Hematopoietic Bone Marrow, hematopoietic stem cells HSCs, immortalizing genes, in vitro redifferentiation of beta cells, nicotinamide, retinoic acid, transducible TAT-Cre, Transiently Immortalized Beta Cells