Self-renewal is a hallmark of stem cells. For many many years, scientists are trying very hard to test self-renewal of mesenchymal stem cells (MSC). But, so far, they were not able to come up with rigorous reproducible self-renewal assay for MSC. As a result, the lack of evidence for self-renewal put into the question of MSC identity and use such terminology as “stem cell”. There are a number of open questions in this ongoing debate. Some of them:
- Should we dismiss the “stem cell” term, based only on failure of self-renewal assays?
- Should serial transplantation assay be applied for MSC or any other adult stem cells? Maybe it’s not required and applied only to hematopoietic stem cells.
- What kind of assays for self-renewal in vivo should be considered or developed for mouse and human MSC?
Self-renewal cannot be reliably proven in vitro (reviewed in Bianco et al. 2008), and the concept of self-renewal became widely confused with the mere ability of a given cell to initiate long-term, extensive proliferation in culture.
Let’s look at some experimental attempts to test MSC self-renewal. Some of them were actually successful. Today, I’ll discuss 3 examples of successful approaches to MSC self-renewal assay.
1. Ectopic transplantable hematopoietic stroma formation in mice
This approach was proposed as the first test for MSC self-renewal more than 30 years ago by Russian scientist Joseph Chertkov. Many of his experiments are summarized in this article.
He was able to serially transplant “hematopoietic stroma” and bone marrow adherent cell layer under renal capsule.
The results show that stromal progenitors capable of hematopoietic microenvironment transfer cannot be transplanted i.v., and do not take part in MSC regeneration after irradiation. On implantation of BM of intact mice or adherent cell layer from bone marrow cultures under the renal capsule, ectopic hematopoietic foci form, in which the hematopoietic cells belong to the recipient while the stroma is of donor origin.
In the each cycle of transplant, hematopoiesis was created de novo from the host in donor-derived stroma. But self-renewal ability was rapidly exhausted:
During the serial transfer of the ectopic hematopoietic tissue without ossicles, a complete loss of the ability to form the hematopoietic focus was already apparent at the third passage. In this case no more than half of the stromal precursors remained on the ossicle (when the bone marrow is pressed out of the femur only 10–15% of the stroma precursors remain on the bone), which was verified by separate implantation of the ossicle and hematopoietic tissue from focus.
After series of published experiments, Chertkov concluded:
The high self-maintenance ability, on the one hand, and the ability to form a fully differentiated bone marrow stromal tissue on the other, suggests that the cells transferring the hematopoietic microenvironment are true mesenchymal stem cells.
Unfortunately, Cherkov is not credited for his fundamental work, which was done more than 30 years ago.
2. Transplantation of clonogenic human CD146+ stromal cells
Paolo Bianco‘s group used sophisticated approach to test self-renewal of human MSC. They identified human bone marrow CD146+ cells as easily assayable clonogenic cells, capable to re-create hematopoietic environment in immunocomromised mice. They were able to isolate the same population of CD146+ cells from heterotopic ossicles and, subsequently, re-establish colonies ex vivo:
We have now shown that following transplantation of CD146+ stromal cells, a small subset retain CD146 expression, dynamically associate with developing sinusoids, and eventually regenerate heterotopic human cells with the anatomy and phenotype of ARCs.
We have also shown that transplantation of cell populations derived from either a limited number of CD146+ CFU-Fs or single CD146+ CFU-Fs results in the re-establishment, in the heterotopic ossicles, of CD146+ CFU-Fs that can be secondarily passaged and directly assayed. By providing evidence for the ability of CD146+ stromal cells to function as self-renewing, clonogenic skeletal progenitors, our data outline the long sought anatomical identity of SSCs (“mesenchymal” stem cells) in human BM and a crucial feature of their phenotype.
3. Transplantation of mouse Nestin+ MSC
The most recent and successful attempt to test MSC self-renewal was undertaken by Paul Frenette‘s group. They isolated Nestin+ bone marrow stromal cells and rigorously test them for “stemness”. The primary Nestin+ mesenspheres were transplanted subcutaneously and gave “hematopoietic ossicles”. After 2 months of transplant, the secondary Nestin+ mesenspheres were isolated from the ossicles and transferred in secondary recipient. Finally, after 8 months of secondary transplant, they were able to isolate Nestin+ spheres from secondary “hematopoietic ossicles”:
Thus, these results demonstrate that nestin+ cells are indeed bona fide MSCs capable of self-renewal in serial transplantations.
Even though, we now have some proofs for MSC self-renewal, the simple and robust assay still missing. Scientists applied the same experimental approach (ectopic serial transplantation) to different populations of bone marrow-derived stromal cells. The Chertkov’s approach looks more promising to me. I think, it could be easily reproduced and combined with colonies formation. It looks very similar to bone marrow transplantation as hematopoietic stem cell assay. Both of them assess “unknown” stem cell populations and clones, but rigorously measure functional activity. What do you think?