Neural induction of adult stem cells in vitro – real thing or artifact?

by Alexey Bersenev on August 9, 2013 · 3 comments

in cell culture, neural

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Many medical researchers believe that neural differentiation of mesenchymal stromal cells (MSC) or cord blood stem cells is a real thing and make a lot of sense for justification of cell therapy clinical trials in neurology. There is a countless number of studies, which demonstrate that MSC could be easily induced into mature neurons. Quick analysis of the literature left me with a lot of questions about those studies and I remain skeptical. Some of my observations:

  1. Conclusions about neuronal differentiation based only on markers expression – phenotypic and genototypic. I’d roughly estimate > 90% of studies, which assess only markers by flow cytometry and gene expression profiling. It typically goes like this: primary MSC –> culture in cocktail of chemicals (neuronal inducers) for 2-4 weeks –> expression of 3-5 neuronal markers and genes –> we got neurons!
  2. Lack of functional assays. Very few studies assess function of adult stem cell-derived neuronal cells. For example, electrophysiology and transplantation assays (integration in the brain and synaptic and metabolic activity in vivo).
  3. Lack of appropriate controls, such as neuronal cell lines, primary freshly isolated neurons and non-induced MSC.
  4. There is no data on reproducibility of protocols. There are many of neural induction protocols, which use a great variety of cell culture techniques and supplements, but all of them (or most of them) are not validated by independent labs.
  5. Despite some controversial data in neural induction of adult stem cells, there is no ongoing discussion about validity of the data and its necessity for clinical translation.

Now, I’m going to give you a few examples. Because there are a lot of studies, which claim generation of neurons or glial cells from MSC or cord blood, I’ll go over some controversial data.

Formation of neurons by non-neural adult stem cells – artifact of growth in culture:

Here we present evidence that shows that cultured bone marrow-derived stem cells express neural proteins and form structures resembling neurons under defined growth conditions. We demonstrate that these changes in cell structure and neural protein expression are not consistent with typical neural development.

Induction of bone marrow stromal cells to neurons as cell culture artifact:

We therefore explored the potential of simple chemical methods to transdifferentiate other cell types, including primary rat fibroblasts, primary human keratinocytes, HEK293 cells, rat PC-12 cells, and as positive control rat bone marrow stromal (BMS) cells. Surprisingly, all cells except for keratinocytes adopted at least partial “neuron-like” pyramidal cell morphology with fine-cellular extensions resembling neurites upon stimulation with BME or DMSO/BHA. However, time-lapse microscopy indicated that the chemical exposure of MSCs did not result in new neurite growth but rather cellular shrinkage, with retraction of the majority of existing cell extensions, leaving only few, fine neurite-like processes.

Neuronal morphology, markers but lack of function:

After 6 h of neuronal induction, the cells had assumed neuronal morphologies and expressed neuron-specific enolase, beta-III-tubulin, neurofilament-H and HNK-1, while only a subpopulation expressed CD15 and synaptophysin. However, electrical signaling could not be detected, neither spontaneously nor after electrical stimulation. Nevertheless, transmission electron microscopy revealed cellular features of neuritogenesis and synaptogenesis…

Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: Disruption of actin cytoskeleton:

Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time-lapse studies. Similar neurite-like processes were induced by treating MSC with DMSO only or with actin filament-depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers.

Reading through papers, I have got even more questions about translation of neural induction into clinical practice:

  1. What is the point therapeutic benefit in neural induction if most of professionals currently pretty sure (with the evidence for it) that neuroprotection from adult stem cells occurs via paracrine mechanisms? There are studies and preliminary results of clinical trials, which demonstrate that MSC and cord blood cells possess neuroprotective properties without any neuronal induction in vitro.
  2. Are there any comparative studies for efficacy of “native” adult stem cells (ex: MSC) versus neuronal-induced? For example, one study demonstrates that umbilical cord-derived MSC can cause re-myelination in animal model without any induction in vitro.
  3. What are evidence for migration, engraftment and differentiation in central nervous system of transplanted induced MSC or cord blood cells?
  4. What is the difference in therapeutic benefit between adult stem cell-derived neurons/ glia and neuronal-induced cells (which retain pre-induced properties)?

I don’t think we have answers to these questions. Nonetheless, many clinical trials have been justified by the assumption that MSC or cord blood cells pre-differentiated in vitro will be therapeutically beneficial. As far as I’m concern, there is no evidence for that from experimental studies.

For example, Kurtzberg’s studies on oligodendrocyte progenitors, derived from cord blood stem cells, do not address the questions I mentioned above. At least they have a function assay:

We observed myelination of shiverer neurons in our functional assay, which could be used as a potency assay for release of OPC cells in phase I human clinical trials.

Yet another study, which tries to justify the use of MSC-derived neural progenitors in multiple sclerosis, fails to explain why we need induction in vitro. In discussion they wrote:

Our analysis with neural-committed human MSC-NPs showed that only a small fraction of MSC-NPs retain adipogenic or osteogenic differentiation potential compared with MSCs, indicating that reduced mesodermal differentiation capacity and increased homogeneity of MSC-NPs may decrease the risk of ectopic differentiation upon CNS transplantation. Indeed, unlike with MSCs, there were no abnormal cellular masses found after intrathecal injection of syngeneic MSC-NPs into EAE mice [15].

If you follow the link they refer to, you will not able to find MSC control versus MSC-NP (neural progenitors) cell therapy model. Seem like nobody compared these 2 cell types properly in cell transplantation model of multiple sclerosis.

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{ 3 comments… read them below or add one }

Bill Busa August 10, 2013 at 8:01 am

Thanks for this article, Alexey. As one whose early work in biology involved electrophysiological recording, I’m constantly amazed at the many papers that claim neural induction of stem cells without bothering to perform the definitive test: stick a couple of microelectrodes in there and test for the ability to conduct an action potential! I do appreciate that relatively few cell biologists are equipped or trained to do electrophysiology, but it’s not rocket surgery!

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Jim H August 11, 2013 at 6:01 pm

I think the question is a bit odd. If we believe iPS cells can be made from MSC and most other adult cell types, one could use iPS to derive neurons from “adult stem cells, ” right?

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Alexey Bersenev August 13, 2013 at 10:41 am

Hi Jim,
I don’t think it’s odd. I clearly stated in the post what i mean under “neural induction of adult stem cells”. Even though, cord blood is not “adult”, I was talking about bone marrow MSC and cord blood stem cells precisely.

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