SCIENCE OF REGENERATION

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HOW NATURE DOES IT

The Axolotl salamander is able to regrow brain and spinal cord tissue as well as entire limbs after injury and tissue loss. It achieves this by direct somatic cell reprogramming rather than recruiting circulating stem cells for regrowing the tissue.

Following injury, the surface of the wound is covered by a “wound epidermis” which is formed from epithelial cells that have migrated to the site of injury. Plasma, tissue fragments, and cellular debris accumulate under the wound epidermis forming a matrix. Dedifferentiated cells are released from the underlying tissue and accumulate at the site of injury, and together with the matrix form a blastema. The “dedifferentiated” cells within the blastema have re-accessed their own respective “cell development” programs for tissue formation, by undergoing reprogramming events to their own respective multipotent state, as if each somatic cell type had a memory of its origin which was triggered by the injury. Regeneration progresses and the multipotent cells divide, migrate and eventually differentiate down their own respective lineage, yielding within 40-50 days a perfectly restored limb or organ, in full respect of the original architecture and with no scar formation.

Fortuna Fix has perfected the same method for direct somatic cell reprogramming into neural stem (or precursor) cells.

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STEM CELLS AND DIRECT REPROGRAMMING

Fortuna Fix's process for direct somatic cell reprogramming into neural precursor cells is based on the Axolotl Salamander’s well documented ability to regenerate.

The cell is the basic building block of all living organisms and the human body is composed of trillions of cells. They provide structure for the body, take in nutrients from food, convert those nutrients into energy, and carry out specialized functions. These functions are determined by the genes that are expressed in that particular cell.

All cells in the human body contain the same genes (~22,000), but only certain ones are “expressed” in each particular cell, making the proteins that determine the functions of that cell.

A group of different cells working together to fulfill a function constitutes a tissue (e.g., neurons-astrocytes-oligodendrocytes working synergistically constitute neuronal tissue). A group of tissues working together to fulfill a bodily function constitutes an organ (e.g., the brain or the spinal cord). Finally, a concerted group of organs are re-grouped in a system (e.g. the brain and spinal cord constitute the Central Nervous System).

If cells make tissues, organs and systems, then what makes cells?

Stages of development: totipotent, pluripotent, and multipotent cells
At the beginning of human life when the sperm fertilizes the egg, totipotent cells are born of the zygote. They possess the ability to become anything (placenta and embryo) and are present in such form up to the 16-cell stage (i.e., within the first couple of cell divisions after fertilization, up to 4 days post fertilisation).

Following this stage of totipotency, as the number of cells increases, pluripotent cells emerge. Pluripotent cells retain the ability to make any cell of the embryo (embryonic stem cells are pluripotent), but lose the ability to become placenta.

After 3 weeks post fertilisation, pluripotent cells begin to organize themselves into 3 germ layers (Ectoderm, Mesoderm, and Endoderm) and progressively differentiate towards a more defined state.

Multipotent stem cells are specialized stem cells differentiated from one of the three germ layers, but restricted within a specific lineage. Each type of multipotent stem cell makes the specialized somatic cells of its lineage: for example, a neural stem cell is a multipotent stem cell that originates in the ectodermal germ layer and is the only multipotent stem cell that has the ability to make neurons, astrocytes and oligodendrocytes (and thus, neuronal tissue), and does not have the ability to form any other tissue.

Direct cell reprogramming is designed to allow a patient’s cell to “jump” directly to the specialized multipotent or unipotent cell of choice, thus tailor-making autologous multipotent or unipotent cells that can differentiate or grow into specific and specialized cell types (e.g., A9 dopaminergic neurons, which are the neurons that are lost and need to be replenished in patients with Parkinson’s disease). 

Fortuna Fix’s reprogramming process is accomplished in vitro within 1-2 weeks by transient expression of reprogramming factors delivered into the patient’s cells (with no use of animal components or viruses and no genetic engineering).

One key feature of this process involves chromatin remodeling, by which the DNA wrapped within nucleosomes becomes accessible to transcription factors and the replication machinery. One of the reprogramming factors releases the chromatin and exposes the cell’s DNA to the other reprogramming factors that drive the reprogramming of the somatic cell to the neural multipotent or unipotent cell by directly triggering the expression of at least one master gene regulator resulting in the expression of a number of secondary genes characteristic of structural and functional properties of the desired neural multipotent or unipotent cells. Stable expression of the master and secondary genes is achieved by allowing the chromatin to remodel and lock into the new neural multipotent or unipotent epigenetic state. Following such a reprogramming event in vitro, the new cells are incubated in reagents and media components chemically optimized for the neural cells.

The resulting directly reprogrammed neural precursor cells (drNPCs) have the ability to continue proliferating for many passages and differentiate along their specific neural, astrocyte and/or oligodendrocyte cell lineage, ultimately resulting in mature functional neurons, astrocytes and/or oligodendrocytes. One of the effects of the direct cell reprogramming is the lengthening and stabilization of the telomeres, with consequent rejuvenation of the reprogrammed cells.

 

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Holy Grail: directly reprogrammed autologous multipotent stem cells
Today, autologous multipotent stem cell therapy is regarded by many as the “Holy Grail” for regenerating and replacing damaged, lost, or aged cells in organs. Bone marrow and fat (adipose) stem cells are effective therapeutic multipotent stem cells for replacing bone, cartilage and fat cells. However, they do not have the ability to replace cells in other organs, where cells are derived from other lineages.

Direct cell reprogramming is uniquely designed to allow a patient’s cell to “jump” directly to the specialized multipotent stem cell of choice, thus tailor-making autologous multipotent stem cells needed to repair a damaged or diseased organ. 

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The process developed by Fortuna Fix is accomplished in vitro within 6-12 days by transient expression of reprogramming factors delivered using a synthetic plasmid (with no use of animal components or viruses and no integration into the genome).

One key feature of this process involves chromatin remodeling, by which the DNA wrapped within nucleosomes becomes accessible to transcription factors and the replication machinery. One of the reprogramming factors releases the chromatin and exposes the cell’s DNA to the other reprogramming factors that drive the reprogramming of the somatic cell to the neuronal stem cell by directly triggering the expression of at least one master or regulator gene resulting in the expression of a number of secondary genes characteristic of functional (and phenotypical) neural stem cells. Stable expression of the master and secondary genes is achieved by allowing the chromatin to remodel and lock into the neural stem cell epigenetic state. Following such a reprogramming event in vitro, the new cells are incubated in reagents and media components chemically optimized for neural stem cells.

 
 

The resulting directly reprogrammed neural precursor cells (drNPCs) differentiate into a neural/glial/oligodendrocyte progenitor cell, ultimately differentiating to a neuron, an astrocyte or an oligodendrocyte. One of the effects of the direct cell reprogramming is the stabilization of the telomeres of the multipotent neural stem cells and the transient expression of telomerase, a clear sign of stemness and with consequent rejuvenation of the reprogrammed cells.

 
 
 
 
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