Mechanisms of therapeutic action in cell therapy – a paradigm shift

by Alexey Bersenev on April 21, 2011 · 5 comments

in under discussion

Post to Twitter

 
Intro

When we apply cell therapy in the clinic we want to injected cells to fix tissue damage/ disease. It is important to know the mechanisms by which cells will be able to repair/ regenerate a tissue.

Historically, the first and the most important mechanism that was proposed, is a cell replacement. It was very neat and nice explanation – irreversibly damaged cells must be replaced by “brand new” normal ones in order to restore a function. The classical example of this – hematopoietic stem cell (HSC) transplantation in hematology-oncology. In leukemic patients, the whole blood is totally replaced by donor-derived cells  after transplant for life. Things got much less clear when people started to apply HSC for “non-hematopoietic diseases” and “non-hematopoietic progenitors” for a variety of conditions without pre-conditioning the patients. In this case cell replacement was not detectable or was not tracked. Nonetheless, many of those clinical trials are showing a benefit for the patients, so elucidating mechanisms is important.

The decade of studying the mechanisms of therapeutic action in cell therapy have brought to us very interesting and significant data. These data shifted the current paradigm (damaged cells must be replaced by donor cells in order to heal) and expanded our understanding of how donor cells can function and interact with a host after transplantation. We can actually witness a few recent paradigm shifts in cell therapy – (1) from organ transplant to cell transplant (can we do it?), (2) from cell replacement to repair host tissue without replacement, (3) from cell transplant to “cell therapy without cells”. I collected some new exciting data on this issue and I’m sharing them below.

Significance

  1. Development of potency assays based on known mechanism of action to allow successful cell product characterization and approval.
  2. Development of new “smart regenerative therapies”. For example,“cell therapy without cells” – modification of endogenous cell properties (migration, adhesion, growth factor release) by exogenous agents (drug or biologic) in order to repair damaged tissue.
  3. Discussion: “Do we need to transplant cells at all?

These data call into question the need for cell transplantation for many types of therapy, in particular for acute injuries such as myocardial infarction and stroke.

The mechanisms

1. Stimulation of endogenous resident progenitor cells
This stimulation could be direct (cell contact required) or indirect (via niche modification or paracrine mechanisms). There are a number of studies, published on this mechanism, but I’d like to point to only two recent ones. Loffredo and others have showed that regenerative effect of cardiac cell therapy could be due to stimulation of resident cardiac progenitors by exogenous injected cells:

Many possible explanations for cell therapy-mediated improvements in cardiac function have been proposed, including direct transdifferentiation of exogenously delivered cells into cardiovascular cells, cell therapy-mediated cardioprotection, and paracrine stimulation of angiogenesis or endogenous progenitors.
The central finding of this study is that cell therapy with c-kit+ bone marrow progenitors after experimental myocardial infarction activates endogenous cardiac progenitors.

Another recent study demonstrates that human autologous bone marrow cell infusion can activate hepatic progenitor cells in patients with liver cirrhosis:

We investigated serial pathological features and the clinical impact after autologous bone marrow infusion (ABMI) in patients with advanced LC. Ten patients with advanced LC due to chronic hepatitis B virus infection underwent ABMI.
ABMI is suggested to improve liver function and to activate the progenitor cell compartment. Although clinical improvement was sustained for more than 6 months, histological changes in the liver returned to baseline 6 months after ABMI. Further comparative studies are warranted.

2. Fusion
The mechanism of bone marrow cell fusion with organ tissue cells as potentially therapeutic is remaining elusive. Even though, therapeutic benefit of fusion was demonstrated in experiement, we still don’t know its significance for clinic.

The discussion is still ongoing, partly because the frequency of fusion events are very rare. It seem to be very hard to evaluate therapeutic value of rare event, separately from other mechanisms. Recently, fusion proposed to be a mechanism of so-called “reprogramming in situ”:

Because stem cells are often found to improve repair tissue including heart without evidence of engraftment or differentiation, mechanisms underlying wound healing are still elusive. Several studies have reported that stem cells can fuse with cardiomyocytes either by permanent or partial cell fusion processes.
We found that heterologous cell fusion promoted cardiomyocyte reprogramming back to a progenitor-like state.
Finally, we showed that stem cell mitochondria were transferred into cardiomyocytes, persisted in hybrids and were required for somatic cell reprogramming.

3. Horizontal material transfer (microvesicles, exosomes, mitochondria)
There is increasing experimental evidence for the modification of host cells by genetic or non-genetic material transferred from transplanted exogenous cells via other than fusion mechanisms. This kind of mechanisms together with fusion could explain such described phenomena as reprogramming in situ or transdifferentiation. Importantly, transfer of biomolecules between cells could be bidirectional – (1) toward host cells (could explain protective or regenerative effect) or (2) toward exogenous donor cells (could explain transdifferentiation). Genetic or non-genetic cargo could be transferred via the following mechanisms:

(A) exosomes
It was shown that mesenchymal stem cell-derived exosomes can cause cardioprotective effects:

These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.

(B) apoptotic bodies
Internationalization of apoptotic bodies is newly described mechanism of horizontal genetic material transfer:

We found that DNA can be horizontally transferred from hematopoietic to epithelial cell lines through phagocytosis of apoptotic bodies.
We propose that the incessant charge of the transplant recipient with donor-DNA and its “inappropriate” intranuclear delivery in host epithelium may explain the emergence of epithelial cells with donor-derived genome.

(C) microvesicles
Microvesicles may transfer genetic and non-genetic cargo, such as receptors, proteins, mRNA and microRNA to target cells. The role of microvesicles in cell therapeutic effects is nicely reviewed here and here and here.
I’d like to note that more and more work has been done with human microvesicles.

(D) mitochondria transfer via intercellular contact
This phenomenon was described in vitro in mesenchymal stem cells and cardiomyocytes co-culture system:

Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.


(F) surface molecules internalization by endosomes via intercellular contact
This phenomenon was described for human hematopoitic cells and osteoblasts:

Authors used in vitro live-cell imaging co-culture system to unveil communications between human HSC and osteoblasts.
The authors demonstrate that at the contact site, containing some signal molecules (CD63, CD133…), portions of this domain was taken up by osteoblasts and internalized into endosomes.

4. Transdifferentiation
I’ve wrote about transdifferentiation before:

We still don’t know what is the role of transdifferentiation/ fusion in disease healing and tissue repair.
Because of the rarity and unknown physiological meaning, the possibility of transdifferentiation can not justify therapeutic potential of infused bone marrow cells. In other words, we can not currently use plasticity/ transdifferentiation magic as a mechanism of action of therapeutic cell products.

For me, transdifferentiation as a therapeutic mechanism has only historical interest. Seem like transdifferentiation was the only one potential cell replacement mechanism without recipient conditioning. But it’s not proven until now.

5. General paracrine stimulation
(A) angiogenesis
Transplanted cells (for example mesenchymal stem cells) could release some angiogenic factors and therefore improve vascularization of ischaemic tissue:

Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.

(B) “trophic factors”
We here investigated the effects exerted by the recently characterized immortalized haematopoietic progenitor cell line DKmix and their conditioned medium in a murine wound healing model.
Abundant levels of matrix metalloproteinase -2 and -9 in the supernatants were detected. Protein arrays of the supernatants revealed a strong secretion of cytokines and growth factors, such as monocyte chemoatractant protein-1 and GM-CSF from DKmix cells.
DKmix cells improve skin-substitute wound healing by promoting angiogenesis as well as migration and proliferation of fibroblasts.

(C) anti-apoptotic effects
These effects were demonstrated for mesenchymal stem cell-derived SDF-1 as possible neuroprotective mechanism in parkinsonian model:

Consequently, MSC transplantation might exert neuroprotection on 6-OHDA-exposed dopaminergic neurons at least partly through anti-apoptotic effects of SDF-1α. The results demonstrate the potentials of intravenous MSC administration for clinical applications, although further explorations are required.

6. Immunological and anti-inflammatory mechanisms
Mesemchymal stem cell are known as potent immunomodulators. They can cause a bunch of immunological effects upon transplantation. Current view on MSC-induced immunomodulation was nicely reviewed here and here.

Very elegant study which was done by Darwin Prockop’s group showed that human MSC improve cardiac function by secretion of anti-inflammatory protein TSG-6.

Concluding remark

Discoveries of the past decade showed that cells after transplant can act in other than engraftment and simple function replacement mechanisms. Unveiling of such mechanisms raises an important question: “Do we need cell transplant to regenerate a damaged tissue?” I think, while our knowledge is still limited about the factors, released by cells, we do need them. Cells could produce a complex variety of factors, naturally mixed in “appropriate” concentrations. Sometimes, transplanted cells need direct contact with host tissue in order to heal. For example, transplanted exogenous neural stem cells can cause neuroprotection of the host neurons only via formation of gap junctions. So, in the future in some indications just a matrix tuned by growth factors could be enough, by in other indications only viable functional cells could help. We need to use all possibilities and play this game in a very smart way.

In conclusion, more and more evidence indicates that exogenous donor cells can heal the tissue by other than cell replacement mechanisms. Finally, two important points:

  • we don’t know what kind of cells can do that, if cell population is not purified, but heterogenous mixture (could be mature cells or progenitors or stem cells);
  • we still don’t understand all of the mechanisms; we frequently can not estimate their therapeutic value; we frequently can not uncouple them from each other.

{ 5 comments… read them below or add one }

Doug Sipp April 24, 2011 at 3:24 am

Hi Alexey – This is great stuff. You’ve summarized quite a lot of recent evidence that points to alternative modes of action for injected non-engrafting cells, and I’m looking forward to going through some of the primary research you’ve cited.

However, I wonder about the viability of using cells, rather than other non-living vehicles, for the delivery of factors that will presumably be subject to degradation/metabolism in the same way other drugs and biologics are. Cells are notoriously expensive, time-conusming, and difficult to work with – it seems that if the objective of injecting cells is some secondary effect, rather than long-term functional engraftment, that there should be less “messy” ways of triggering it. I understand your points about complex cocktails of secreted factors, and cell-cell signaling issues, such as gap junctions, but these too are reducible problems that can presumably be addressed by research, which would allow for better quality control, reliability in dosage, purity, etc. than can be achieved using populations of cells.

Reply

Alexey Bersenev April 25, 2011 at 1:26 am

Doug,
Thank you for comment!
I completely agree with you. Undefined mechanisms of cell populations in mix can give us more troubles in terms of (1) cell product characterization (potency assay) from developers point of view, (2) therefore regulation hurdles, (3) inappropriate justification of using “whole marrow” or “whole cord blood” by bogus commercial cell therapy clinics and marketers.
If we only know, what cell type is really matter and what is a mechanism (factor) which make this cell type work therapeutically in human body, our lives will be much easier. My view is that as soon as we will figure out the mechanisms (other than replacement-engraftment), we will able to replace cell therapy by more sophisticated, less laborious, less expensive, less “messy” approaches. I actively promote approaches like “cell therapy without cells” and “cell therapy in situ”. I’m sure that we will see more and more of these kind of approaches in play. We will figure out more and more mechanisms. This review is just a first attempt to summarize some experimental data. Bear in mind – we really don’t know anything yet about the mechanisms in human body.

Reply

Doug Sipp April 25, 2011 at 5:32 am

Thanks Alexey – a clear, fair, and accurate assessment! As always.

Reply

Aline M Betancourt April 26, 2011 at 12:22 pm

Hi Alexey, I appreciate all of your postings and comments. I just wanted to add that our group more recently reviewed the immune modulating properties of MSCs in SCRT: Stem Cell Res Ther. 2010 Nov 11;1(5):34.PMID:21092149. Thanks, Ali

Reply

Alexey Bersenev April 28, 2011 at 1:03 am

Aline,
Thank you for update! Prockop’s group work inspired me to write this.

Reply

Leave a Comment

Previous post:

Next post: