Olfactory bulb neural progenitor cells transferred to pancreas express β cell genes and produce insulin.





Scientists say adult neural progenitor cells taken from the brains of diabetic mice are capable of producing insulin when transplanted back into the pancreas of diabetic animals. The animal studies demonstrated that neural stem cells grafted into the pancreas were triggered to express pancreatic beta-cell genes and generated enough insulin to lead to significantly reduced blood glucose levels in diabetic animals.

Reporting in EMBO Molecular Medicine, the team, led by Makoto Asashima, Ph.D., and Tomoko Kuwabara, at Japan’s National Institute of Advanced Industrial Science and Technology (AIST), claim the technique didn’t need the introduction of any inductive genes and confirms that there is a strong functional similarity between adult neurons and β cells. Their paper is titled “Insulin biosynthesis in neuronal progenitors derived from adult hippocampus and the olfactory bulb.”

There are numerous similarities between mammalian genes expressed in the developing brain and pancreas, the researchers note. Indeed, previous research has indicated that insulin-producing neurons in the fruit fly are functional analogues of vertebrate pancreatic islet β cells and that both developed from ancestral insulin-producing neurons. “The gene-expression programs for developing neurons and β cells are remarkably similar,” they add.

Moreover, insulin is one of a number of factors that regulates cell fate in hippocampal adult neural stem cells. Diabetes in mice can impair hippocampal learning and memory; in humans, diabetes has been found to increase the risk of clinical depression and dementia.

Mammalian neurogenesis occurs throughout adulthood in the subventricular zone (SVZ) of the forebrain lateral ventricles and in the hippocampus (HPC). NSCs can be obtained from the relatively accessible olfactory bulb. Neurogenesis is triggered by Wnt3 released from astrocytes, and the team’s previous work had demonstrated that adult hippocampal Wnt/beta-catenin signaling triggers the expression of NeuroD1, a transcription factor that plays a key role in the developing brain and pancreas. Studies had also shown that neuroD1 deficiency in mice causes severe diabetes and is lethal because the protein is needed for insulin gene expression.

The investigators are now reporting on a research program to evaluate whether NSCs could generate insulin-secreting cells following transplantation into the pancreas of diabetic animals. They first demonstrated that insulin-1 mRNA is expressed in the neuronal layers of the adult hippocampus but not in layers where astrocytes and undifferentiated NSCs reside. ELISA and immunohistochemical (IHC) analysis also confirmed that the insulin-1 protein was endogenously produced in the adult brain and hippocampus: insulin-producing β-tubulin III-expressing cells were clearly detected in granule neurons in the dentate gyrus, which houses adult HPC self-renewing NSCs. Neurons in the CA1 pyramidal region, cortex, and substantia nigra were also positive for insulin production.

In the brain, astrocytes define the niche that supports neuronal differentiation and secrete Wnt3 factors that promote adult neurogenesis. Interestingly, when the researchers examined α cells in pancreatic tissue, they also detected cells that expressed the astrocyte marker glial fibrillary acidic protein (GFAP). These cells co-localized with Wnt3+ cells, “indicating that α cells release the neurogenic Wnt3 as do hippocampal astrocytes,” they remark.

In contrast, there was a marked reduction in Wnt3+ cells in the pancreas of streptozotocin (STZ)-induced diabetic rats and type II diabetic Goto-Kakizaki (GK) rats. The reduction in Wnt3+ cells in diabetic pancreas tissue was accompanied by reductions in insulin, together with downregulation of Wnt3 mRNA in both pancreatic islets and the HPC, and upregulation of the Wnt inhibitor IGFBP-4.

The observation that neurons can express insulin prompted the researchers to see whether adult NSCs could be used—without requiring any exogenous genetic induction—as a source of insulin-producing cells to treat diabetes. They focused on HPC NSCs, which have been studied extensively, and on OB NSCs, because they are easily accessible.

Culturing cells derived from both tissues under neuron differentiation conditions led to gene expression changes including the downregulation of NSC marker genes and upregulation of neuronal marker gene expression. However, insulin-1 and insulin-2 expression also increased extensively in both HPC and OB neurons.

Microarray analysis suggested that neither HPC nor OB neurons expressed factors such as MafA, Pdx1, or neurogenin 3, which are necessary to activate insulin gene expression in pancreatic beta cell lineages. Quantitative real-time RT-PCR analysis, meanwhile, confirmed that insulin-1 mRNA induction correlated with NeuroD1 mRNA up-regulation in both HPC and OB cells.

This indicates that NeuroD1 expression is required for insulin expression in adult NSCs, in vitro at least, the team notes. OB NSCs and HPC NSCs also both responded similarly and in a dose- dependent manner to Wnt3a ligands.

Despite the positive data generated thus far in vitro, it still wasn’t clear whether adult NSCs would survive and potentially produce insulin within the pancreas. Although the researchers’ studies had confirmed that Wnt3 was produced in pancreatic α cells, it didn’t necessarily mean that adult NSCs could respond to the Wnt3 signals when transplanted into the pancreas.

To evaluate this the team transplanted adult NSCs transduced with Sox2CreGFP (retrovirus-encoding Sox2 promoter-driven Cre/GFP) into the pancreas of animals from the Rosa-GFP mouse line, to trace the fate of transplanted cells. Interestingly, and despite the earlier microarray analyses demonstrating that adult NSCs didn’t normally express pancreatic β cells markers such as MafA, the transplanted Sox2CreGFP+ cells expressed both MafA and pancreas transcription factor 1a (Ptf1a).

This suggested “that adult NSCs possessed the intrinsic ability to express these β cell-specific markers and that their expression levels were modulated by extracellular factors,” the authors remark. Ptf1a is normally required to secure the lineage commitment of endocrine progenitors during embryonic development.

In contrast, when Sox2CreGFP-transduced conditional β-catenin knockout NSCs were tansplanted into the pancreas, there was an evident decline in the numbers of GFP+ cells, and those Sox2CreGFP+ cells that were detected didn’t express insulin. Marker-positive cells with NeuroD1, PTF1a, and MafA were also rare in the β-catenin conditional knockout NSC transplants.

Importantly, there was evidence that the these transplanted NSC cells underwent more frequent apoptosis, “indicating that Wnt/b-catenin signaling in Sox2+ NSCs was important for their survival,” the authors note.

The ultimate test was to see whether NSCs could actually treat diabetes in vivo. Initial tests suggested that the process of microinjecting adult HPC NSCs led to increased levels of apoptosis, so the team instead prepared the cells on collagen sheets which could then be stacked and grafted onto the pancreas. HPC and OB NSCs were prepared from STZ-induced type I diabetic rats and type II diabetic animals.

Because NSCs in diabetic animals had been found to contain higher IGFBP-4 and lower Wnt3 levels than wild-type animals, treating the cultured cells with Wnt3a adn anti-IGFBP-4 rescued insulin expression. The resulting neural progenitor cells were then tagged with a CAG promoter-driven EGFP expression vector so they could be traced following grafting.

The NP cell-carrying collagen sheets were subsequently transplanted back into the pancreas of eight week old rats with the same diabetes type—for example, the cells cultured from type 1 diabetic rats were transplanted back into type 1 diabetic rats 17 weeks after transplantation recipient animals were injected daily with BrdU to detect cell proliferation.

Encouragingly, mice receiving either the HPC- or OB-derived neural progenitor (NP) transplants demonstrated reduced blood glucose levels and upregulated plasma and pancreatic insulin levels. These animals were able to efficiently clear glucose, and GFP+ cells expressing insulin were labelled with BrdU, confirming the cells were proliferating. Removing the grafted collagen sheets from the recipient animals 15–19 weeks after transplantation led to their blood glucose levels rising again.

“These data indicated that regulated insulin secretion by the grafted adult NPs from DB HPC and OB contributed to glucose homeostasis in the host DB rats,” the authors write. “Our present study revealed that adult HPC and OB NSCs possess the intrinsic ability to generate insulin-producing cells via Wnt/b-catenin signalling. We further determined that the best source of insulin supply for transplantation into DB animals was adult NPs that had been committed into early neuronal lineage and had been treated with anti-IGFBP4 and Wnt3a to rescue their impaired Wnt signalling in DB animals.”

They point out the study provides an example of the direct use of adult stem cells from one organ to another, without introducing inductive genes. “Using adult NSCs for treating diabetes is potentially advantageous because donors are not required, the introduction of inductive genes is not necessary, and extracellular and endogenous regulation suitably resembles that employed by adult islet endocrine cell lineages.” And while further validation studies will obviously be necessary, the team points out that “the basic strategy outlined here should be useful for preliminary treatment of diabetes using the patient’s own NSCs before the disease progresses.”



























