MRI/SPECT/Fluorescent Tri‐Modal Probe for Evaluating the Homing and Therapeutic Efficacy of Transplanted Mesenchymal Stem Cells in a Rat Ischemic Stroke Model

Quantitatively tracking engraftment of intracerebrally or intravenously transplanted stem cells and evaluating their concomitant therapeutic efficacy for stroke has been a challenge in the field of stem cell therapy. In this study, first, an MRI/SPECT/fluorescent tri‐modal probe (¹²⁵I‐fSiO4@SPIOs) is synthesized for quantitatively tracking mesenchymal stem cells (MSCs) transplanted intracerebrally or intravenously into stroke rats, and then the therapeutic efficacy of MSCs delivered by both routes and the possible mechanism of the therapy are evaluated. It is demonstrated that ⁽¹²⁵⁾I‐fSiO4@SPIOs have high efficiency for labeling MSCs without affecting their viability, differentiation, and proliferation capacity , and found that 35% of intracerebrally injected MSCs migrate along the corpus callosum to the lesion area, while 90% of intravenously injected MSCs remain trapped in the lung at 14 days after MSC transplantation. However, neurobehavioral outcomes are significantly improved in both transplantation groups, which are accompanied by increases of vascular endothelial growth factor, basic fibroblast growth factor, and tissue inhibitor of metalloproteinases‐3 in blood, lung, and brain tissue (p < 0.05). The study demonstrates that ¹²⁵I‐fSiO4@SPIOs are robust probe for long‐term tracking of MSCs in the treatment of ischemic brain and MSCs delivered via both routes improve neurobehavioral outcomes in ischemic rats.     

An NIR‐II Fluorescence/Dual Bioluminescence Multiplexed Imaging for In Vivo Visualizing the Location,Survival, and Differentiation of Transplanted Stem Cells

The in vivo distribution, viability, and differentiation capability of transplanted stem cells are vital for the therapeutic efficacy of stem cell–based therapy. Herein, an NIR‐II fluorescence/dual bioluminescence multiplexed imaging method covering the visible and the second near‐infrared window from 400 to 1700 nm is successfully developed for in vivo monitoring the location, survival, and osteogenic differentiation of transplanted human mesenchymal stem cells (hMSCs) in a calvarial defect mouse model. The exogenous Ag₂S quantum dot–based fluorescence imaging in the second near‐infrared window is applied for visualizing the long‐term biodistribution of transplanted hMSCs. Endogenous red firefly luciferase (RFLuc)‐based bioluminescence imaging (BLI) and the collagen type 1 promoter–driven Gaussia luciferase (GLuc)‐based BLI are employed to report the survival and osteogenic differentiation statuses of the transplanted hMSCs. Meanwhile, by integrating the three imaging channels, multiple dynamic biological behaviors of transplanted hMSCs and the promotion effects of immunosuppression and the bone morphogenetic protein 2 on the survival and osteogenic differentiation of transplanted hMSCs are directly observed. The novel multiplexed imaging method can greatly expand the capability for multifunctional analysis of the fates and therapeutic capabilities of the transplanted stem cells, and aid in the improvement of stem cell–based regeneration therapies and their clinical translation.     

Multifunctional Upconversion Nanoparticles for Dual‐Modal Imaging‐Guided Stem Cell Therapy under Remote Magnetic Control

Stem cells have generated a great deal of excitement in cell‐based therapies. Here, a unique class of multifunctional nanoparticles (MFNPs) with both upconversion luminescence (UCL) and superparamagnetic properties is used for stem cell research. It is discovered that after being labeled with MFNPs, mouse mesenchymal stem cells (mMSCs) are able to maintain their viability and differentiation ability. In vivo UCL imaging of MFNP‐labeled mMSCs transplanted into animals is carried out, achieving ultrahigh tracking sensitivity with a detection limit as low as ≈10 cells in a mouse. Using both UCL optical and magnetic resonance (MR) imaging approaches, MFNP‐labeled mMSCs are tracked after being intraperitoneally injected into wound‐bearing mice under a magnetic field. The translocation of mMSCs from the injection site to the wound nearby the magnet is observed and, intriguingly, a remarkably improved tissue repair effect is observed as the result of magnetically induced accumulation of stem cells in the wound site. The results demonstrate the use MFNPs as novel multifunctional probes for labeling, in vivo tracking, and manipulation of stem cells, which is promising for imaging guided cell therapies and tissue engineering.     

Tracking of Transplanted Human Mesenchymal Stem Cells in Living Mice using Near‐Infrared Ag2S Quantum Dots

Stem cell therapeutics has emerged as a novel regenerative therapy for tissue repair in the last decade. However, dynamically tracking the transplanted stem cells in vivo remains a grand challenge for stem cell‐based regeneration medicine in full understanding the function and the fate of the stem cells. Herein, Ag₂S quantum dots (QDs) in the second near‐infrared window (NIR‐II, 1.0–1.4 μm) are employed for dynamically tracking of human mesenchymal stem cells (hMSCs) in vivo with high sensitivity and high spatial and temporal resolution. As few as 1000 Ag₂S QDs‐labeled hMSCs are detectable in vivo and their fluorescence intensity can maintain up to 30 days. The in situ translocation and dynamic distribution of transplanted hMSCs in the lung and liver can be monitored up to 14 days with a temporal resolution of 100 ms. The in vivo high‐resolution imaging indicates the heparin‐facilitated translocation of hMSCs from lung to liver as well as the long‐term retention of hMSCs in the liver contribute to the treatment of liver failure. The novel NIR‐II imaging offers a possibility of tracking stem cells in living animals with both high spatial and temporal resolution, and encourages the future clinical applications in imaging‐guided cell therapies.     

A Patch of Detachable Hybrid Microneedle Depot for Localized Delivery of Mesenchymal Stem Cells in Regeneration Therapy

Mesenchymal stem cells (MSCs) have been widely used for regenerative therapy. In most current clinical applications, MSCs are delivered by injection but face significant issues with cell viability and penetration into the target tissue due to a limited migration capacity. Some therapies have attempted to improve MSC stability by their encapsulation within biomaterials; however, these treatments still require an enormous number of cells to achieve therapeutic efficacy due to low efficiency. Additionally, while local injection allows for targeted delivery, injections with conventional syringes are highly invasive. Due to the challenges associated with stem cell delivery, a local and minimally invasive approach with high efficiency and improved cell viability is highly desired. In this study, a detachable hybrid microneedle depot (d‐HMND) for cell delivery is presented. The system consists of an array of microneedles with an outer poly(lactic‐co‐glycolic) acid shell and an internal gelatin methacryloyl (GelMA)‐MSC mixture (GMM). The GMM is characterized and optimized for cell viability and mechanical strength of the d‐HMND required to penetrate mouse skin tissue is also determined. MSC viability and function within the d‐HMND is characterized in vitro and the regenerative efficacy of the d‐HMND is demonstrated in vivo using a mouse skin wound model.     

Protamine Functionalized Single‐Walled Carbon Nanotubes for Stem Cell Labeling and In Vivo Raman/Magnetic Resonance/Photoacoustic Triple‐Modal Imaging

Stem cells have shown great potential in regenerative medicine and attracted tremendous interests in recent years. Sensitive and reliable methods for stem cell labeling and in vivo tracking are thus urgently needed. Here, a novel approach to label human mesenchymal stem cells (hMSCs) with single‐walled carbon nanotubes (SWNTs) for in vivo tracking by triple‐modal imaging is presented. It is shown that polyethylene glycol (PEG) functionalized SWNTs conjugated with protamine (SWNT‐PEG‐PRO) exhibit extremely efficient cell entry into hMSCs, without affecting their proliferation and differentiation. The strong inherent resonance Raman scattering of SWNTs is used for in vitro and in vivo Raman imaging of SWNT‐PEG‐PRO‐labeled hMSCs, enabling ultrasensitive in vivo detection of as few as 500 stem cells administrated into mice. On the other hand, the metallic catalyst nanoparticles attached on nanotubes can be utilized as the T2‐contrast agent in magnetic resonance (MR) imaging of SWNT‐labeled hMSCs. Moreover, in vivo photoacoustic imaging of hMSCs in mice is also demonstrated. The work reveals that SWNTs with appropriate surface functionalization have the potential to serve as multifunctional nanoprobes for stem cell labeling and multi‐modal in vivo tracking.     

Advanced Functional Biomaterials for Stem Cell Delivery in Regenerative Engineering and Medicine

Stem cell–based therapies can potentially regenerate many types of tissues and organs, thereby providing solutions to a variety of diseases and injuries. However, acute cell death, uncontrolled differentiation, and low functional engraftment yields remain critical obstacles for clinical translation. Advanced functional biomaterial scaffolds that can deliver stem cells to the targeted tissues/organs and promote stem cell survival, differentiation, and integration to host tissues may potentially transform the clinical outcome of stem cell–based regenerative therapies. In this review, the authors briefly summarize sources of stem cells for transplantation, present the current state of the art in biomaterial design for stem cell delivery, and provide critical analysis for existing materials. Applications to the cardiovascular, neural, and musculoskeletal systems are highlighted with recent nonclinical studies and clinical trials. The authors also discuss how advances in biomaterials research can contribute to regenerative medicine research and stem cell therapies.     

Multimodal Label-Free Monitoring of Adipogenic Stem Cell Differentiation Using Endogenous Optical Biomarkers

Stem cell-based therapies carry significant promise for treating human diseases. However, clinical translation of stem cell transplants for effective treatment requires precise non-destructive evaluation of the purity of stem cells with high sensitivity (<0.001% of the number of cells). Here, a novel methodology using hyperspectral imaging (HSI) combined with spectral angle mapping-based machine learning analysis is reported to distinguish differentiating human adipose-derived stem cells (hASCs) from control stem cells. The spectral signature of adipogenesis generated by the HSI method enables identifying differentiated cells at single-cell resolution. The label-free HSI method is compared with the standard techniques such as Oil Red O staining, fluorescence microscopy, and qPCR that are routinely used to evaluate adipogenic differentiation of hASCs. HSI is successfully used to assess the abundance of adipocytes derived from transplanted cells in a transgenic mice model. Further, Raman microscopy and multiphoton-based metabolic imaging is performed to provide complementary information for the functional imaging of the hASCs. Finally, the HSI method is validated using matrix-assisted laser desorption/ionization-mass spectrometry imaging of the stem cells. The study presented here demonstrates that multimodal imaging methods enable label-free identification of stem cell differentiation with high spatial and chemical resolution.     

Photoacoustic Imaging of Embryonic Stem Cell‐Derived Cardiomyocytes in Living Hearts with Ultrasensitive Semiconducting Polymer Nanoparticles

Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) have become promising tools to repair injured hearts. To achieve optimal outcomes, advanced molecular imaging methods are essential to accurately track these transplanted cells in the heart. In this study, it is demonstrated for the first time that a class of photoacoustic nanoparticles (PANPs) incorporating semiconducting polymers (SPs) as contrast agents can be used in the photoacoustic imaging (PAI) of transplanted hESC‐CMs in living mouse hearts. This is achieved by virtue of two benefits of PANPs. First, strong photoacoustic (PA) signals and specific spectral features of SPs allow PAI to sensitively detect and distinguish a small number of PANP‐labeled cells (2000) from background tissues. Second, the PANPs show a high efficiency for hESC‐CM labeling without adverse effects on cell structure, function, and gene expression. Assisted by ultrasound imaging, the delivery and engraftment of hESC‐CMs in living mouse hearts can be assessed by PANP‐based PAI with high spatial resolution (≈100 µm). In summary, this study explores and validates a novel application of SPs as a PA contrast agent to track labeled cells with high sensitivity and accuracy in vivo, highlighting the advantages of integrating PAI and PANPs to advance cardiac regenerative therapies.     

Multimodal Magnetic Nanoclusters for Gene Delivery,Directed Migration,and Tracking of Stem Cells

This study develops multimodal magnetic nanoclusters (M‐MNCs) for gene transfer, directed migration, and tracking of human mesenchymal stem cells (hMSCs). The M‐MNCs are designed with 5 nm iron oxide nanoparticles and a fluorescent dye (i.e., Rhodamine B) in the matrix of the Food and Drug Administration approved polymer poly(lactide‐co‐glycolide) using a nanoemulsion method. The synthesized M‐MNCs have a hydrodynamic diameter of ≈150 nm, are internalized by stem cells via endocytosis, and deliver genes with high efficiency. The cellular internalization and gene expression efficiency of the clustered nanoparticles are significantly higher than that of single nanoparticles. The M‐MNC‐labeled hMSCs migrate upon application of a magnetic force and can be visualized by both optical and magnetic resonance (MR) imaging. In animal models, the M‐MNC‐labeled hMSCs are also successfully tracked using optical and MR imaging. Thus, the M‐MNCs not only allow the efficient delivery of genes to stem cells but also the tracking of cells in animal models. Taken together, the results show that this new type of nanocomposite can be of great help in future stem cell research and in the development of cell‐based therapeutic agents.     

Stem Cell-Niche Engineering via Multifunctional Hydrogel Potentiates Stem Cell Therapies for Inflammatory Bone Loss

Effective therapies capable of simultaneously inhibiting inflammation and promoting bone healing remain to be developed for inflammatory bone disease. Stem cell therapies hold great promise for a variety of diseases, but their translation is hampered by low cell survival, rapid clearance, and limited functional integration of transplanted stem cells in target tissues. Herein, a multifunctional hydrogel-based stem cell niche engineering strategy is reported for the treatment of inflammatory bone loss. By rationally integrating different functional modules, an injectable hydrogel-based stem niche is engineered, which possesses temperature-triggered gelling performance, inflammation/oxidative stress-resolving activity, stem-cell binding and survival-enhancing capacity, and osteogenesis-promoting capability. Using ectomesenchymal stem cells (EMSCs), effectiveness of this functionally advanced synthetic stem cell niche is demonstrated in rats with periodontitis, a representative inflammatory bone loss disease. Synergistic effects of the multifunctional hydrogel and EMSCs are also confirmed, with respect to normalizing the pathological microenvironment and improving alveolar bone regeneration in the periodontal tissue. Mechanistically, inflammation/oxidative stress-resolving and osteogenic differentiation promoting capacities of the synthetic stem cell niche are mainly achieved by an incorporated nanotherapy via the GDF15/Atf3/c-Fos axis of the MAPK signaling pathway. Besides periodontitis, the newly engineered hydrogel-stem cell therapies are promising for the treatment of other inflammatory bone defects.     

Nanocarrier‐Mediated Codelivery of Small Molecular Drugs and siRNA to Enhance Chondrogenic Differentiation and Suppress Hypertrophy of Human Mesenchymal Stem Cells

Cartilage loss is a leading cause of disability among adults, and effective therapy remains elusive. Human mesenchymal stem cells (hMSCs), which have demonstrated self‐renewal and multipotential differentiation, are a promising cell source for cartilage repair. However, the hypertrophic differentiation of the chondrogenically induced MSCs and resulting tissue calcification hinders the clinical translation of MSCs for cartilage repair. Here, a multifunctional nanocarrier based on quantum dots (QDs) is developed to enhance chondrogenic differentiation and suppress hypertrophy of hMSCs simultaneously. Briefly, the QDs are modified with β‐cyclodextrin (β‐CD) and RGD peptide. The resulting nanocarrier is capable of carrying hydrophobic small molecules such as kartogenin in the hydrophobic pockets of conjugated β‐CD to induce chondrogenic differentiation of hMSCs. Meanwhile, via electrostatic interaction the conjugated RGD peptides bind the cargo siRNA targeting Runx2, which is a key regulator of hMSC hypertrophy. Furthermore, due to the excellent photostability of QDs, hMSCs labeled with the nanocarrier can be tracked for up to 14 d after implantation in nude mice. Overall, this work demonstrates the potential of our nanocarrier for inducing and maintaining the chondrogenic phenotype and tracking hMSCs in vivo.     

Injectable Hydrogels with In Situ Double Network Formation Enhance Retention of Transplanted Stem Cells

Stem cell transplantation via direct injection is a minimally invasive strategy being explored for treatment of a variety of injuries and diseases. Injectable hydrogels with shear moduli <50 Pa can mechanically protect cells during the injection process; however, these weak gels typically biodegrade within 1–2 weeks, which may be too fast for many therapeutic applications. To address this limitation, an injectable hydrogel is designed that undergoes two different physical crosslinking mechanisms. The first crosslinking step occurs ex vivo through peptide‐based molecular recognition to encapsulate cells within a weak gel that provides mechanical protection from injection forces. The second crosslinking step occurs in situ to form a reinforcing network that significantly retards material biodegradation and prolongs cell retention time. Human adipose‐derived stem cells are transplanted into the subcutaneous space of a murine model using hand‐injection through a 28‐gauge syringe needle. Cells delivered within the double‐network hydrogel are significantly protected from mechanical damage and have significantly enhanced in vivo cell retention rates compared to delivery within saline and single network hydrogels. These results demonstrate that in situ formation of a reinforcing network within an already existing hydrogel can greatly improve transplanted cell retention, thereby enhancing potential regenerative medicine therapies.     

3D‐Printed Microrobotic Transporters with Recapitulated Stem Cell Niche for Programmable and Active Cell Delivery

Poor retention rate, low targeting accuracy, and spontaneous transformation of stem cells present major clinical barriers to the success of therapies based on stem cell transplantation. To improve the clinical outcome, efforts should focus on the active delivery of stem cells to the target tissue site within a controlled environment, increasing survival, and fate for effective tissue regeneration. Here, a remotely steerable microrobotic cell transporter is presented with a biophysically and biochemically recapitulated stem cell niche for directing stem cells towards a pre‐destined cell lineage. The magnetically actuated double‐helical cell microtransporters of 76 µm length and 20 µm inner cavity diameter are 3D printed where biological and mechanical information regarding the stem cell niche are encoded at the single‐cell level. Cell‐loaded microtransporters are mobilized inside confined microchannels along computer‐controlled trajectories under rotating magnetic fields. The mesenchymal stem cells are shown retaining their differentiation capacities to commit to the osteogenic lineage when stimulated inside the microswimmers in vitro. Such a microrobotic approach has the potential to enable the development of active microcarriers with embedded functionalities for controlled and precisely localized therapeutic cell delivery.     

In Situ Crosslinkable Gelatin Hydrogels for Vasculogenic Induction and Delivery of Mesenchymal Stem Cells

Clinical trials utilizing mesenchymal stem cells (MSCs) for severe vascular diseases have highlighted the need to effectively engraft cells and promote pro‐angiogenic activity. A functional material accomplishing these two goals is an ideal solution as spatiotemporal and batch‐to‐batch variability in classical therapeutic delivery can be minimized, and tissue regeneration would begin rapidly at the implantation site. Gelatin may serve as a promising biomaterial due to its excellent biocompatibility, biodegradability, and non‐immuno/antigenicity. However, the dissolution of gelatin at body temperature and quick enzymatic degradation in vivo have limited its use thus far. To overcome these challenges, an injectable, in situ crosslinkable gelatin was developed by conjugating enzymatically crosslinkable hydroxyphenyl propionic acid (GHPA). When MSCs are cultured in 3D in vitro or injected in vivo in GHPA, spontaneous endothelial differentiation occurs, as evidenced by marked increases in endothlelial cell marker expressions (Flk1, Tie2, ANGPT1, vWF) in addition to forming an extensive perfusable vascular network after 2‐week subcutaneous implantation. Additionally, favorable host macrophage response is achieved with GHPA as shown by decreased iNOS and increased MRC1 expression. These results indicate GHPA as a promising soluble factor‐free cell delivery template which induces endothelial differentiation of MSCs with robust neovasculature formation and favorable host response.     

Ice‐Templated Protein Nanoridges Induce Bone Tissue Formation

Little is known about the role of biocompatible protein nanoridges in directing stem cell fate and tissue regeneration due to the difficulty in forming protein nanoridges. Here an ice‐templating approach is proposed to produce semi‐parallel pure silk protein nanoridges. The key to this approach is that water droplets formed in the protein films are frozen into ice crystals (removed later by sublimation), pushing the surrounding protein molecules to be assembled into nanoridges. Unlike the flat protein films, the unique protein nanoridges can induce the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts without any additional inducers, as well as the formation of bone tissue in a subcutaneous rat model even when not seeded with MSCs. Moreover, the nanoridged films induce less inflammatory infiltration than the flat films in vivo. This work indicates that decorating biomaterials surfaces with protein nanoridges can enhance bone tissue formation in bone repair.     

Bright Aggregation‐Induced Emission Dots for Dynamic Tracking and Grading of Patient‐Derived Xenografts in Zebrafish

Cancer prognosis will benefit from a scoring system that could grade malignant traits of patient‐derived cells by assessing their growth and metastasis in a living system. Specific tracking of patient‐derived cells requires labeling by contrast agents with good signal‐to‐noise ratio and no specific stain of host tissues. Towards this aim, aggregation‐induced emission (AIE) dots are developed for in vivo cancer tracking with emphasis on reproducible optimized formulation and specific fluorescent labeling of cells that enable enhanced spatial temporal resolution in vivo. The importance of energy‐dependent AIE dots uptake for patient‐derived cell labeling is emphasized to reveal their specific uptake by viable cancer cells. Using optically transparent zebrafish embryo, the ability is demonstrated to follow the engraftment of transplanted AIE dot labeled cells in zebrafish brains over one week. Cells detected outside the brain after 7 d are quantified as metastatic cells. Results from seven clinical samples demonstrate the utility of this methodology to differentiate low engraftment level of benign neoplasms from higher engraftment level and metastasis detected in malignant ovarian cancer specimens. Achieving clinically validated results supports the use of AIE dot labeled patient derived cells in zebrafish xenografts for future cancer prognosis.     

In Vivo Micro‐CT Imaging of Human Mesenchymal Stem Cells Labeled with Gold‐Poly‐l‐Lysine Nanocomplexes

Developing in vivo cell tracking is an important prerequisite for further development of cell‐based therapy. So far, few computed tomography (CT) cell tracking studies have been described due to its notoriously low sensitivity and lack of efficient labeling protocols. A simple method is presented to render human mesenchymal stem cells (hMSCs) sufficiently radiopaque by complexing 40 nm citrate‐stabilized gold nanoparticles (AuNPs) with poly‐l ‐lysine (PLL) and rhodamine B isothiocyanate (RITC). AuNP‐PLL‐RITC labeling does not affect cellular viability, proliferation, or downstream cell differentiation into adipocytes and osteocytes. Labeled hMSCs can be clearly visualized in vitro and in vivo with a micro‐CT scanner, with a detection limit of ≈2 × 10⁴ cells per µL in vivo. Calculated Hounsfield unit values are 2.27 per pg of intracellular Au, as measured with inductively coupled plasma mass spectrophotometry, and are linear over a wide range of cell concentrations. This linear CT attenuation is observed for both naked AuNPs and those that were taken up by hMSCs, indicating that the number of labeled cells can be quantified similar to the use of radioactive or fluorine tracers. This approach for CT cell tracking may find applications in CT image‐guided interventions and fluoroscopic procedures commonly used for the injection of cellular therapeutics.     

Bright and Photostable Organic Fluorescent Dots with Aggregation‐Induced Emission Characteristics for Noninvasive Long‐Term Cell Imaging

Efficient long‐term cell tracing in a noninvasive and real‐time manner is of great importance to understand genesis, development, invasion, and metastasis of cancerous cells. Cell penetrating organic dots with aggregation‐ induced emission (AIE) characteristics are successfully developed as long‐term cell trackers. The AIE dots enjoy the advantages of high emission efficiency, large Stokes shift, good biocompatibility, and high photostability, which ensure their good performance in long‐term non‐invasive in vitro cell tracing. Moreover, it is the first report that AIE dots exhibit certain permeability to cellular nucleus, making them attractive potential candidates for nucleus imaging. The AIE dots display superior performance compared to their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for monitoring biological processes.