Drawbacks of current reprogramming technologies for rejuvenation purpose:
iPS technology is able to rejuvenate somatic cells. However, it also involves complete dedifferentiation, i.e. somatic cell identities were lost after they were reprogrammed to pluripotent state. As a result, iPS cells can't be used directly in human body, as it can cause tumor formation. In other words, iPS cells will have to be differentiated in vitro to a certain type of somatic cell type before being used in human body.​
Current achievement on rejuvenation:
Partial reprogramming:
An important approach is partial reprogramming. 2-5 days partial reprogramming is a method of using Yamanaka factors (or alternative reprogramming factors, in the wider context) to revert aged cells to a younger state without completing the reprogramming cycle, thus retaining their cellular identity. However, partial reprogramming still face the challenge of partial dedifferentiation of the somatic cells (though it was claimed the dedifferentiation is reversable) and limited rejuvenation capability.
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Clearance of senescent cells:
Using senolytic, among a class of small molecules that can selectively induce death of senescent cells and improve health in humans. Studies in BubR1 progeroid mice provided proof-of-principle that clearance of senescent cells can delay age-related degenerative pathologies16.
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Overexpression of the pluripotency factor NANOG: ​
Overexpression of the pluripotency factor NANOG in progeroid or senescent myogenic progenitors reversed cellular aging and fully restored their ability to generate contractile force. The effect was mediated by the reactivation of the ROCK and TGF-β pathways.
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3 factors for in vivo rejuvenation
Using the eye as a model CNS tissue, that ectopic expression of Oct4, Sox2, and Klf4 (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. (Lu Y, Brommer B, Tian X, Krishnan A, Meer M, Wang C, et al. Reprogramming to recover youthful epigenetic information and restore vision. Nature. 2020;588(7836):124-9.)
What we are doing:
iPS reprogramming is a highly regulated process controlled by the orchestrated interaction of a vast gene network. This network likely consists of two overlapping and entangled sub-networks—one responsible for dedifferentiation and the other for rejuvenation. To rejuvenate cells without inducing dedifferentiation, we need to selectively activate genes involved in rejuvenation while inhibiting genes linked to dedifferentiation. Identifying these genes, however, is an extremely complex challenge.
Since iPS reprogramming technology is currently the only approach capable of rejuvenating somatic cells (along with dedifferentiation) using a small set of defined factors—such as Yamanaka factors (OSKM genes) or modified gene combinations—we use iPS technology as a starting point. We believe that OSKM genes and other related factors sit at the top of a vast regulatory gene network. More specifically, six genes—Oct4, Klf4, Sox2, Myc, Lin28, and Nanog (OKSMLN)—have been proven to efficiently reprogram cells into iPSCs, positioning them as key regulators of the reprogramming process.
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Our Approach
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We leverage our proprietary artificial intelligence model to analyze downstream gene networks associated with the OKSMLN factors. By examining gene expression databases and scientific literature, we identify candidate genes that may promote rejuvenation or suppress dedifferentiation. These candidates are then used to design various gene combinations, which undergo further screening.
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To facilitate this process, we have developed a proprietary in vitro rejuvenation screening platform. This platform allows us to efficiently screen, identify, and characterize gene combinations that rejuvenate senescent cells while maintaining their original identity.
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Our work is an ongoing process, and we continuously refine and optimize our gene combinations to improve the efficiency and precision of cellular rejuvenation.
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Making iPS Cells
Rejuvenation strategy-1
Rejuvenation strategy-2
What we have achieved?
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By Using these advanced tools, we have identified a gene rejuvenation cocktail that successfully rejuvenates senescent human artery endothelial cells (HAECs) while preserving their original cell phenotype. (U.S. Patent Pending, Application #63412420 and #63415286).
Figure 1
Figure 1: characterization of young and senescent human artery endothelial cells (HAEC).
Characterization of young (pX+d4.56) and senescent (pX+d12.97) HAECs.
As expected, pX+d4.56 HAECs are small and exhibit typical endothelial morphology under a light microscope. They are β-Gal negative, indicating that they are not senescent, as confirmed by their continuous growth capability. Additionally, they are CD31 and VWF positive, confirming their endothelial identity.
In contrast, pX+d12.97 HAECs appear larger while retaining endothelial morphology under a light microscope. However, they are β-Gal positive, signifying cellular senescence, as further evidenced by their lack of proliferative capacity. Despite senescence, these cells remain CD31 and VWF positive, confirming that they still maintain their endothelial identity. ​
Figure 2.
Figure 2. Rejuvenation of Senescent HAECs Using Our Gene Cocktail
Following treatment with our gene cocktail, senescent HAECs exhibit morphology similar to young endothelial cells under a light microscope. They are β-Gal negative, indicating the loss of senescence, and their restored growth capability confirms their rejuvenated state.
Additionally, the treated cells remain CD31 and VWF positive, demonstrating that they have retained their endothelial identity. These findings confirm that our gene combination successfully rejuvenates senescent HAECs while preserving their original cell phenotype.
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Figure 3.
Figure 3. Doubling Times (n) of Rejuvenated HAECs Before Growth Cessation
The rejuvenated HAECs treated with our gene combination were continuously cultured, and their doubling times (d) were calculated. The rejuvenated endothelial cells exhibited doubling times of approximately 22–24 before reaching senescence again.
The initial rejuvenation screening was conducted on senescent HAECs (pX+d12.97), where the senescent cells had an unknown passage number (pX) with an additional 12.97 doublings (d12.97). After rejuvenation, the cells regained proliferative capability, completing an additional 22–24 doublings before ceasing growth once more.
These results demonstrate that rejuvenated cells retain a limited proliferative capacity, confirming that they are non-tumorigenic and do not exhibit uncontrolled growth.