By Jacob Pawlak, Biochemistry and Molecular Biology ’23

Author’s Note: I wrote this piece to bring attention to the exciting new field of research being conducted primarily in China that aims to control the differentiation of stem cells by irradiating them with different wavelengths of light. This non-invasive method is potentially of great value to those working in regenerative medicine and has a strong foundation of research for future exploration. I hope to introduce this fascinating concept to future researchers to pique their interest in the field. 

Introduction 

Regenerative medicine is the use of human bodily mechanisms to restore functionality, cure diseases, and heal injuries. In particular, this field’s research is primarily concerned with healing previously untreatable injuries to the nervous system and regrowing musculoskeletal tissue from the aftereffects of traumatic injury or disease. The body is incapable of repairing traumatic injury to the brain or spinal cord completely, and regenerative medicine aims to use stem cells as a source of new tissue. This primarily consists of manipulating stem cells into specific cell types at the injury sites. These injuries are not particularly uncommon; in just the US, an average of 17,000 people a year are hospitalized for spinal cord damage [1].

Within the field of regenerative medicine, remote control of stem cell differentiation is one of its most promising areas of research, as it avoids the major issue of complications arising from invasive procedures, such as a risk for infection or an autoimmune response. Non-invasiveness is typically achieved through the use of near-infrared light, which is capable of penetrating tissue layers to reach target sites without harming normal cells in its path [2]. Invasive procedures require large surgical teams and expose the patient to potential infection. Instead, non-invasive procedures can be done as part of outpatient care, not requiring lengthy hospital visits. Photobiomodulation has recently emerged as one of the most promising candidates for remote control of stem cell differentiation. Photobiomodulation is the act of exposing cells to specific wavelengths of light to influence gene expression and shift cellular processes towards a specific target, such as increased differentiation into a target cell type or increased proliferation of the stem cells [2]. This often takes the form of the exposed light influencing crucial biochemical pathways in the cells, “pushing” the cells towards the researcher’s target cell type or towards increased replication. Upon reaching the site, the photons can influence the cell’s genetic expression on its own, or be converted into other influential wavelengths by novel optical devices we will go on to describe in this review [2]. 

There are two primary cell types targeted by researchers in regenerative medicine. The first are neural stem cells, which are directed to differentiate into astrocytes, cells that act to regulate blood flow and repair the nervous system following infections and injuries [3]. The second target for control are mesenchymal stem cells, which are found throughout the body and are capable of differentiating into a wide variety of musculoskeletal cell types such as bone, muscle, and  cartilage [4]. These two cell types form the backbone of photobiomodulation research due to their potential use in regenerating complex, irreparably damaged organs such as the spinal cord.  

This review presents the findings of recent research into the remote control of human stem cell differentiation. While these researchers have not worked with in vivo cells, they have laid the groundwork for a wide range of potential exploration routes for further research into stem cell differentiation control via photobiomodulation and novel optical methods. 

Types of Stem Cells Researched

The two primary types of stem cells being researched for remote control are mesenchymal stem cells and neural stem cells. Mesenchymal stem cells are typically selected to create bone cell cultures, while neural stem cells are directed towards forming glial cells, which support the nervous system by forming sheaths around neural pathways and regulating blood flow [3,4]. 

Mesenchymal stem cells have been the primary focus of novel research, due to the relative ease of acquiring human-adipose derived stem cells (hADSC). These cells are extracted from human fat tissue and are capable of differentiation into multiple cell types. Most papers have focused on increasing the proliferation of these cells alongside increasing their differentiation into osteoblasts [7-9]. These cells are primarily useful in regenerative medicine for application to traumatic injuries to the musculoskeletal system.  

In both types of cells, the light triggers photoreceptor complexes that are sensitive to the upper and lower bound of visible light wavelengths, or red and blue light [5-9]. These complexes induce the cellular modifications that lead to the changes in the stem cell’s rates of proliferation and differentiation. Thus, research focuses primarily on only red, blue, and occasionally green light, as stem cells are not uniquely reactive to more moderate colors on the visible light spectrum due to lack of sufficient sensitivity.  

Novel Non-Invasive Methods – Photobiomodulation

Typically in photobiomodulation research, LED diodes are placed over cell cultures to irradiate them at specific light wavelengths for approximately 60 minutes daily over the course of 5-10 days [5-8]. Once this period is complete, researchers examine the cell cultures for signs that the cells have differentiated, such as the release of signature proteins into the culture medium or through visual inspection with a microscope [5-8].  

In 2019 Wang et al. was successful in multiplying the proliferation of neural stem cells by 4.3x, and their differentiation rate into astrocytes by 2.7x through treatment with low-power blue light irradiation [5]. Proliferation measures the rate of population increase of cells, while differentiation measures how many of the stem cells develop into specialized cells. A newer study in 2021 by Yoon et al. found an increase in astrocyte proliferation through red-light treatment [6]. Notably, Yoon was able to find that red light could influence astrocyte proliferation without affecting other cells in the area, demonstrating the light’s effects in an environment closer to the human body, unlike Wang’s work on isolated astrocyte cultures. These papers making use of different wavelengths of light for different situations speaks to the versatility of photobiomodulation as a method of controlling astrocyte populations. 

Comparing Light Wavelengths 

While some innovative work has been done with novel optical devices, most research deals with the cheaper and simpler direct application of visible light. Visible light has been applied to both neural and mesenchymal stem cells to observe their reactions and to find wavelengths that can control differentiation and proliferation of these cells.  

The application of visible light can be broadly split into two categories: red and blue. Exclusively red-light wavelengths have been found to increase the proliferation of both  mesenchymal and neural stem cells [6, 8-9]. Meanwhile, blue light has mixed effects. On neural stem cells, it increases proliferation and differentiation into astrocytes, whereas on mesenchymal stem cells it has been found to lower proliferation while raising the rate of differentiation into osteoblasts [5, 7-8]. Some preliminary work has been done on green light, which has been found to cause the same effects as blue light on mesenchymal stem cells, due to the two colors’ close proximity on the visible light spectrum [7].

The most promising results come from the work of Crous et al., whose team found that they could increase both differentiation and proliferation in mesenchymal stem cells by alternating red and green light irradiation, synthesizing the effects of the two wavelengths [9]. In combination with Wang Y et al.’s work on mesenchymal stem cells with single wavelength application, this suggests that the inhibitory effects of green light on proliferation are less potent than the enhancing effects of red light, and may even be overwritten completely. This alternation between lower energy red light and higher energy green light poses the clearest path forward for future research into direct light application to stem cells, as increasing both effects is synergistic for tissue regeneration and injury repair. 

Novel Non-Invasive Methods – Upconversion Nanoparticles (UCNPs)

While most research is conducted exclusively with the application of light, novel optical devices have been developed to work in conjunction with light application for finer control of stem cell differentiation. Upconversion nanoparticles (UCNPs) are artificial, nano-scaled lattices of various metal ions that exhibit the capacity to upconvert photons, a process that involves absorbing two lower-energy photons and releasing them as a single higher-energy photon [1, 13]. These are especially useful in regenerative medicine research as they are easily taken in by cells [13]. Wang K. et al and Zhang Y.  et al. both used UCNPs in conjunction with near-infrared (NIR) light to control stem cell  differentiation [11-12]. Both teams irradiated their cell cultures with NIR light, which can penetrate deeper than higher-energy light. This NIR light then activated UCNPs within the cultures to release UV light that activated stem cell differentiation factors from where they were loaded onto the UCNPs. Wang’s team was able to increase mesenchymal stem cell osteogenic differentiation, while Zhang’s team successfully observed increased differentiation into glial cells, a broad category which includes astrocytes and other nervous system support cells.  

Additionally, UCNPs are not exclusively used to release differentiation factors, as Wang M et al.’s team was able to use UCNPs to upconvert NIR light into visible blue light to achieve deeply penetrating visible light exposure, which has high potential for use at injury sites [5]. The use of UCNPs can circumvent the problems of direct application of UV light to cell cultures, such as genetic damage and low tissue penetration. These UCNPs could provide a tool by which stem cells can be influenced in more selective regions, as their area of effect is limited to tissue sites where they have been directly implanted. 

However, while UCNPs have utility, they still pose a challenge to future regenerative medicine research in that they must be somehow applied directly to the target stem cells, requiring an invasive method such as injections. This is a common issue that different research teams have run into. A novel alternative to UCNPs has been developed by Zhang S. et al that applies NIR light to copper sulfide nanostructures [10]. These nanostructures produce electromagnetic oscillations upon stimulation with NIR light that have been found to increase the differentiation of hADSCs into neuron-like cells. While innovative, this method still requires the invasive placement of copper-sulfide nanomaterials at the site of target stem cells to impact their differentiation. Although it provides a potential alternative to UCNPs, this method has not been tested by any other research teams on stem cell cultures and requires a great deal of further research before it can be implemented as a regenerative medicine procedure. Regardless of the nanobiology tools selected, researchers still must identify a way to place their developed structures near target cells. 

Future Possibilities

A potential future for photobiomodulation research lies in the combination of Wang M et al.’s work with UCNPs in combination with visible light application and the alternating light method of Crous et al. to achieve increased proliferation and differentiation with relatively non-invasive deep tissue injury site access [5, 9]. 

Additionally, Crous’ work with earlier neural photobiomodulation studies gives researchers a way to potentially further increase astrocyte proliferation and differentiation by combining Wang M. et al.’s blue light method with Yoon SR et al.’s red light method, thereby activating two different gene expression pathways simultaneously [5-6]. 

The alternating light method requires further inquiry, but Crous et al. have delivered promising results in the form of increased cell movement towards a specific direction in their alternating light research [9]. This directionality is important for regenerative medicine, as cells need to be directed to grow and differentiate at specific points in injury sites to prevent undesired cell growth that could interfere with normal tissue function. Directional application of green and red light could be used in specific patterns to direct mesenchymal stem cells to grow towards a  target site. Specific control over the shape and structure of stem cell differentiation is the next  step for regenerative medicine research, as it allows for the construction of more complicated  tissues and structures for larger injuries and for potential use in organ regeneration. 

Ultimately, this research is based on the application of light; the specifics of potential applications can still be tweaked. As previously mentioned, LED diodes are placed to irradiate samples for roughly an hour a day for approximately a week [5-8]. Scientists have prioritized similar methods to achieve comparable results with each other, but the use of such regular conditions leaves photobiomodulation open for a great deal of further experimentation, as  optimized application of visible light has not yet been determined. Longer or shorter exposure  times, alongside lowering or raising the power of the light sources has not yet been attempted on  stem cell cultures. Furthermore, work with direct light has not been performed on cells that are  heavily obscured from the light source, as would be expected at the site of a deeply placed  traumatic injury in a clinical setting.  

Once photobiomodulation has been optimized and readied for clinical use, it is likely that further work will be performed on not just cell cultures, but on in vivo stem cells and 3D structures of stem cells. In vivo refers to cells experimented on in live organisms, rather than in isolated cultures. In vivo stem cells come with the problem of difficult to control environmental conditions, but they more accurately simulate tissue conditions. Given additional advancements in bioprinting technology, stem cells in 3D structures like hydrogels have the potential for applications of light at different angles. However,  a wide array of new environmental variables would require many years of preparatory work to make up for a lack of current research. Theoretically, it may be possible for a 3D printed structure of stem cells in hydrogels to be selectively irradiated with light to grow more complex tissue formations. As such, photobiomodulation has several avenues for future research to explore as biotechnology  advances. 

Conclusion 

The remote control of stem cells has seen great advancements in the past five years, with  research on novel optical devices, a variety of wavelengths, cutting-edge manipulation methods,  and the production of two different categories of human stem cells. The field has even more potential in the future, especially through the combination of different research team’s work, through synthesizing the use of different wavelengths of light, UCNPs, and currently unrealized advances in biotechnology and nanotechnology. The concept of applying light to cells to control them is an appealing one, and the field will likely expand as methods become more standardized and easier to implement in molecular biology labs. The past five years of research have laid a solid foundation for future research into remote control of stem cell differentiation, and future advances may grant regenerative medicine immensely useful tools for treating traumatic injuries to the nervous system and musculoskeletal system.

References:

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