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Abstract Tuberculosis (TB) is the top cause of death from a single infectious pathogen after COVID-19. Despite molecular diagnostic advances, two-thirds of the 10 million annual TB cases are still diagnosed using direct smear microscopy which has ~50% sensitivity. To increase the analytical performance of smear microscopy, we developed and characterized a novel polymer (Polydiallyldimethylammonium chloride [PDADMAC]) engraftment on inexpensive polystyrene (PS) specifically functionalized for mycobacterial capture. Engraftment is achieved via UV photopolymerization of DADMAC monomer on plasma-activated PS. The platform was tested on sputum from presumptive TB cases in Kampala, Uganda (n = 50), with an increased overall sensitivity of 81.8% (27/33) vs. fluorescent smear microscopy 57% (19/33) compared to a molecular (Cepheid GeneXpert MTB/RIF) gold standard. Frugal smear diagnostic innovation that is rapid and does not require dedicated instrumentation may offer an important solution to bridge the TB diagnostic gap. Graphical Abstract

The transfection efficiency and stability of the delivery vehicles of plasmid DNA (pDNA) are critical metrics to ensure high-quality and high-yield production of viral vectors. We previously identified that the optimal size of pDNA/poly(ethylenimine) (PEI) transfection particles is 400–500 nm and developed a bottom-up assembly method to construct stable 400-nm pDNA/PEI particles and benchmarked their transfection efficiency in producing lentiviral vectors (LVVs). Here, we report scale-up production protocols for such transfection particles. Using a two-inlet confined impinging jet (CIJ) mixer with a dual syringe pump set-up, we produced a 1-L batch at a flow rate of 100 mL/min, and further scaled up this process with a larger CIJ mixer and a dual peristaltic pump array, allowing for continuous production at a flow rate of 1 L/min without a lot size limit. We demonstrated the scalability of this process with a 5-L lot and validated the quality of these 400-nm transfection particles against the target product profile, including physical properties, shelf and on-bench stability, transfection efficiency, and LVV production yield in both 15-mL bench culture and 2-L bioreactor runs. These results confirm the potential of this particle assembly process as a scalable manufacturing platform for viral vector production.

Peripheral nerve injuries remain a challenging problem in need of better treatment strategies. Despite best efforts at surgical reconstruction and postoperative rehabilitation, patients are often left with persistent, debilitating motor and sensory deficits. There are currently no therapeutic strategies proven to enhance the regenerative process in humans. A clinical need exists for the development of technologies to promote nerve regeneration and improve functional outcomes. Recent advances in the fields of tissue engineering and nanotechnology have enabled biomaterial scaffolds to modulate the host response to tissue repair through tailored mechanical, chemical, and conductive cues. New bioengineered approaches have enabled targeted, sustained delivery of protein therapeutics with the capacity to unlock the clinical potential of a myriad of neurotrophic growth factors that have demonstrated promise in enhancing regenerative outcomes. As such, further exploration of combinatory strategies leveraging these technological advances may offer a pathway towards clinically translatable solutions to advance the care of patients with peripheral nerve injuries. This review first presents the various emerging bioengineering strategies that can be applied for the management of nerve gap injuries. We cover the rationale and limitations for their use as an alternative to autografts, focusing on the approaches to increase the number of regenerating axons crossing the repair site, and facilitating their growth towards the distal stump. We also discuss the emerging growth factor-based therapeutic strategies designed to improve functional outcomes in a multimodal fashion, by accelerating axonal growth, improving the distal regenerative environment, and preventing end-organs atrophy.

Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics but assessing the mRNA packaging characteristics in LNPs is challenging. Here, the authors report that mRNA and lipid contents in LNP formulations can be quantitatively examined by multi-laser cylindrical illumination confocal spectroscopy at the single-nanoparticle level.

Plasmid DNA offers extended transgene expression duration compared to mRNA technologies. Here, using a multi-step screening platform, the authors report the best performing nanoparticle formulations for liver-targeted plasmid DNA expression in vivo.

Summary: Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for CNS demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination-promoting compounds, we generated a genome-engineered human pluripotent stem cell (hPSC) line that consists of three reporters: identification-and-purification tag, GFP, and secreted-NanoLuc, driven by the endogenous PDGFRA, PLP1, and MBP genes, respectively. Using this cell line, we established a high-throughput drug screening platform and performed a small-molecule screen, which identified at least two myelination-promoting small-molecule (Ro1138452 and SR2211) that target prostacyclin (IP) receptor and retinoic acid receptor-related orphan receptor γ (RORγ), respectively. Single-cell-transcriptomic analysis of differentiating OPCs treated with these molecules further confirmed that they promote oligodendrocyte differentiation and revealed several pathways that are potentially modulated by them. The molecules and their target pathways provide promising targets for the possible development of remyelination-based therapy for MS and other demyelinating disorders.

Summary: Cholangiocarcinoma (CCA) is a biliary tree malignancy with a dismal prognosis. Tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs) has been shown to be involved in drug resistance. To model the interactions between cancer cells and the TME, we established CCA complex patient-derived organoids (cPDOs) to include epithelial PDO (ePDOs) and matched CAFs. While ePDOs were sensitive to bortezomib, we found the matched cPDOs were relatively resistant. Mechanistically, this resistance was correlated with over-expression of CXCR4 in the CAF component of cPDOs. In accord with the role of CXCR4 in the resistance to bortezomib, we found that a CXCR4 inhibitor can reverse the resistance to bortezomib in vivo. Furthermore, we found that the inhibition of CXCR4 allowed bortezomib to sensitize CCA to anti-PD1 treatment, with a significant reduction of tumor burden and long-term overall survival. This novel cancer/stroma/immune triple treatment holds great promise for the treatment of CCA.

Abstract Treatment of cancers in the lung remains a critical challenge in the clinic for which gene therapy could offer valuable options. We describe an effective approach through systemic injection of engineered polymer/DNA nanoparticles that mediate tumor-specific expression of a therapeutic gene, under the control of the cancer-selective progression elevated gene 3 (PEG-3) promoter, to treat tumors in the lungs of diseased mice. A clinically tested, untargeted, polyethylenimine carrier was selected to aid rapid transition to clinical studies, and a CpG-free plasmid backbone and coding sequences were used to reduce inflammation. Intravenous administration of nanoparticles expressing murine single-chain interleukin 12, under the control of PEG-3 promoter, significantly improved the survival of mice in both an orthotopic and a metastatic model of lung cancer with no marked symptoms of systemic toxicity. These outcomes achieved using clinically relevant nanoparticle components raises the promise of translation to human therapy.

Brain myelinating oligodendrocytes are rare and difficult to isolate, which has limited data on their development. Here the authors develop a reporter for scalable purification of human pluripotent stem cell derived oligodendrocyte lineage cells, and use this to map differentiation using single cell RNA-sequencing,

Tissue engineering strategies that combine human pluripotent stem cell-derived myogenic progenitors (hPDMs) with advanced biomaterials provide promising tools for engineering 3D skeletal muscle grafts to model tissue development in vitro and promote muscle regeneration in vivo. We recently demonstrated (i) the potential for obtaining large numbers of hPDMs using a combination of two small molecules without the overexpression of transgenes and (ii) the application of electrospun fibrin microfiber bundles for functional skeletal muscle restoration following volumetric muscle loss. In this study, we aimed to demonstrate that the biophysical cues provided by the fibrin microfiber bundles induce hPDMs to form engineered human skeletal muscle grafts containing multinucleated myotubes that express desmin and myosin heavy chains and that these grafts could promote regeneration following skeletal muscle injuries. We tested a genetic PAX7 reporter line (PAX7::GFP) to sort for more homogenous populations of hPDMs. RNA sequencing and gene set enrichment analyses confirmed that PAX7::GFP-sorted hPDMs exhibited high expression of myogenic genes. We tested engineered human skeletal muscle grafts derived from PAX7::GFP-sorted hPDMs within in vivo skeletal muscle defects by assessing myogenesis, engraftment and immunogenicity using immunohistochemical staining. The PAX7::GFP-sorted groups had moderately high vascular infiltration and more implanted cell association with embryonic myosin heavy chain (eMHC) regions, suggesting they induced pro-regenerative microenvironments. These findings demonstrated the promise for the use of PAX7::GFP-sorted hPDMs on fibrin microfiber bundles and provided some insights for improving the cell–biomaterial system to stimulate more robust in vivo skeletal muscle regeneration.

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Patients who sustain peripheral nerve injuries (PNIs) are often left with debilitating sensory and motor loss. Presently, there is a lack of clinically available therapeutics that can be given as an adjunct to surgical repair to enhance the regenerative process. Insulin-like growth factor-1 (IGF-1) represents a promising therapeutic target to meet this need, given its well-described trophic and anti-apoptotic effects on neurons, Schwann cells (SCs), and myocytes. Here, we review the literature regarding the therapeutic potential of IGF-1 in PNI. We appraised the literature for the various approaches of IGF-1 administration with the aim of identifying which are the most promising in offering a pathway toward clinical application. We also sought to determine the optimal reported dosage ranges for the various delivery approaches that have been investigated.

A bone marrow-derived mesenchymal stromal cell (MSC) transplant and a bioengineered nanofiber-hydrogel composite (NHC) have been shown to stimulate nervous tissue repair in the contused spinal cord in rodent models. Here, these two modalities were combined to assess their repair effects in the contused spinal cord in adult rats. Cohorts of contused rats were treated with MSC in NHC (MSC-NHC), MSC in phosphate-buffered saline (MSC-PBS), NHC, or PBS injected into the contusion site at 3 days post-injury. One week after injury, there were significantly fewer CD68+ cells in the contusion with MSC-NHC and NHC, but not MSC-PBS. The reduction in CD86+ cells in the injury site with MSC-NHC was mainly attributed to NHC. One and eight weeks after injury, we found a greater CD206+/CD86+ cell ratio with MSC-NHC or NHC, but not MSC-PBS, indicating a shift from a pro-inflammatory towards an anti-inflammatory milieu in the injury site. Eight weeks after injury, the injury size was significantly reduced with MSC-NHC, NHC, and MSC-PBS. At this time, astrocyte, and axon presence in the injury site was greater with MSC-NHC compared with MSC-PBS. We did not find a significant effect of NHC on MSC transplant survival, and hind limb function was similar across all groups. However, we did find fewer macrophages at 1 week post-injury, more macrophages polarized towards a pro-regenerative phenotype at 1 and 8 weeks after injury, and reduced injury volume, more astrocytes, and more axons at 8 weeks after injury in rats with MSC-NHC and NHC alone compared with MSC-PBS; these findings were especially significant between rats with MSC-NHC and MSC-PBS. The data support further study in the use of an NHC-MSC combination transplant in the contused spinal cord.

Purpose: Insulin-like growth factor 1 (IGF-1) is a potent mitogen with the potential to enhance axonal regeneration and minimize muscle atrophy and Schwann cell senescence following prolonged denervation after peripheral nerve injury. IGF-1 is a small protein with a half-life of 5 min, making local delivery a challenge. Our group has demonstrated over 6 weeks of sustained release of bioactive IGF-1 encapsulated within biodegradable nanoparticles (NP) and subsequently developed a nanofiber fiber hydrogel composite (NHC) carrier to retain IGF-1 NPs at target tissue locally for the duration of drug release. The aim of this study was to further characterize and refine the IGF-1 NP-NHC drug delivery and then investigate its efficacy in both rodent and non-human primate (NHP) median nerve injury models. Methods: IGF-1 was encapsulated in biodegradable PCL NPs and then embedded within the NHC composed of hyaluronic acid and PCL nanofibers. Release kinetics and biocompatibility were evaluated and optimized both in vitro and in vivo. The drug delivery system was assessed using a chronic denervation median nerve injury rat model and an acute median nerve repair NHP model. IGF-1NP/NHC was injected along the median nerve and within denervated muscle. In rodents, a range of IGF-1 doses (300, 900 and 1500 μg/mL) were investigated to evaluate dose-response relationships. Axonal regeneration, muscle atrophy, neuromuscular junction reinnervation and recovery of grip strength were assessed. Results: The refined NP-NHC delivery system provided sustained release of bioactive IGF-1, in vivo, for at least 42 days by serial ELISA. IGF-1 treated rodents demonstrated a 35% increase in functional recovery (stimulated grip strength) compared to untreated rodents, with no differences observed between the different concentrations of IGF-1 that were evaluated. Median nerve histomorphometry demonstrated a significantly greater total number of axons at each concentration of IGF-1 compared to untreated rodents (p

Purpose: Fractionated fat (FF) has been shown to promote dermal regeneration; however, the use of fat grafting for reconstruction of soft tissue defects is limited due to volume loss over time. We have developed a novel approach for engineering of vascularized soft tissue using an injectable nanofiber hydrogel (NHC) enriched with FF. Methods: FF was generated by emulsification of groin fat pads from rats and mixed in a 3:1 ratio with NHC (NHC-FF). NHC-FF or NHC alone were placed into isolation chambers together with arteriovenous (AV) loops, which were subcutaneously implanted into the groin of rats (n=8 per group). After 21 days, animals were euthanized, systemically perfused with ink, and tissue was explanted for histological analysis. Immunofluorescent staining (IF) and confocal laser scanning microscopy were used to quantify CD34+ progenitor cells and macrophage subpopulations. Results: NHC-FF tissue maintained its shape without shrinking and showed a significantly stronger functional neovascularization compared to NHC alone at 21 days after implantation (mean vessel count: 833.5 ± 206.1 vs. 296.5 ± 114.1, p = 0.04). Tissue remodeling and cellular infiltration were greater in NHC-FF (mean cell count: 49,707 ± 18,491 vs. 9,263 ± 3,790, p = 0.005) with a significantly higher amount of progenitor cells and regenerative CD163+ macrophages compared to NHC alone. Conclusion: FF-enriched NHC transforms into highly vascularized soft tissue over 21 days without signs of shrinking and promotes macrophage polarization toward regenerative phenotypes. Enrichment of injectable NHC with FF represents a promising approach for durable reconstruction of soft tissue defects.

Due to their ability to provide efficient mixing at small scales, confined impinging jet mixers (CIJMs) are employed widely in nanoparticle assembly processes such as flash nanoprecipitation and flash nanocomplexation, which require rapid mixing. In this mixing device, two jets from opposite directions impinge directly on each other forming a thin shear layer that breaks down rapidly into small flow structures. This enables effective mixing of the species transported by each jet by drastically reducing the diffusion distance. In the present study, the mixing performance of a commonly used cylindrical CIJM is examined by direct numerical simulations. Analysis of the simulation results indicates that the interaction of the shear layer with the inner walls of the CIJM is critical in inducing a range of instabilities in the impinging jet flow. By examining flow structures, statistical quantities, and metrics, we have characterized and quantified the mixing quality of a binary mixture in the CIJM. Product uniformity in processes such as precipitation and complexation is expected to depend on the residence time of the constituents, and this quantity is also calculated and compared for the cases with different jet Reynolds numbers. The jet Reynolds numbers of Re = 200, 600, and 1000 are considered, and the simulation results show that the CIJM achieves very good mixing for the Re = 600 and Re = 1000 cases. It is also found that the Re = 600 case performs slightly better than the other cases in terms of uniformity of the residence time. These quantitative analyses offer useful insights into the mechanism of nanoparticle size control and uniformity afforded by the unique flow physics and mixing characteristics in the CIJMs.

Cell-free DNA (cfDNA) released from damaged or dead cells can activate DNA sensors that exacerbate the pathogenesis of rheumatoid arthritis (RA). Here the authors use ~40 nm cationic nanoparticles to scavenge cfDNA, and demonstrate the potential for nanomedicine to relieve debilitating RA symptoms.

Sustained immune responses, particularly antibody responses, are key for protection against many endemic infectious diseases. Antibody responses are often accompanied by T helper (Th) cell immunity. Herein we study small biodegradable poly (ethylene glycol)-b-poly (lactic-co-glycolic acid) nanoparticles (PEG-b-PLGA NPs, 25–50 nm) as antigen- or adjuvant-carriers. The antigen carrier function of PEG-b-PLGA NPs was compared against an experimental benchmark polystyrene nanoparticles (PS NPs, 40–50 nm), both conjugated with the model antigen ovalbumin (OVA-PS NPs, and OVA-PEG-b-PLGA NPs). The OVA-PEG-b-PLGA NPs induced sustained antibody responses to Day 120 after two immunizations. The OVA-PEG-b-PLGA NPs as a self-adjuvanting vaccine further induced IL-4 producing T-helper cells (Th2), but not IFN-γ producing T-cells (Th1). The PEG-b-PLGA NPs as a carrier for CpG adjuvant (CpG-PEG-b-PLGA NPs) were also tested as mix-in vaccine adjuvants comparatively for protein antigens, or for protein-conjugated to PS NPs or to PEG-b-PLGA NPs. While the addition of this adjuvant NP did not further increase T-cell responses, it improved the consistency of antibody responses across all immunization groups. Together these data support further development of PEG-b-PLGA NPs as a vaccine carrier, particularly where it is desired to induce Th2 immunity and achieve sustained antibody titers in the absence of affecting Th1 immunity.

Current treatment options for volumetric muscle loss (VML) are limited due to donor site morbidity, lack of donor tissue, and insufficient functional recovery. Tissue-engineered skeletal muscle grafts offer the potential to significantly improve functional outcomes. In this study, we assessed the potential pro-myogenic effects of human adipose-derived stem cells (ASCs) seeded onto electrospun uniaxially aligned fibrin hydrogel microfiber bundles. Although both uninduced and 5-azacytidine-induced ASCs exhibited alignment, elongation, and diffuse muscle marker expression when grown on microfiber bundles for 2 months in vitro , both groups failed to fully recapitulate myotube characteristics. To assess the muscle regeneration potential of ASCs in vivo , ASC-seeded fibrin microfiber bundles were implanted in a robust murine VML defect model. Minimal fibrosis was observed surrounding implanted acellular hydrogel fibers at 2 and 4 weeks, and fibers seeded with ASCs exhibited up to 4 times higher volume retention than acellular fibers. We observed increased numbers of cells positive for the regenerating muscle marker embryonic myosin and the mature muscle marker myosin heavy chain in ASC-seeded fibers compared with acellular fibers at 1 and 3 months post-transplantation. Regenerating muscle cells were closely associated with ASC-derived cells and in some cases had potentially fused with them. These findings demonstrate that despite failing to undergo myogenesis in vitro , ASCs combined with electrospun fibrin microfibers moderately increased muscle reconstruction in vivo compared with acellular fibers following a severe VML defect.

Schwann cells are key players in peripheral nerve regeneration, and are uniquely capable of remyelinating axons in this context. Schwann cells orchestrate this process via a set of transcription factors. While it has been shown that overexpression of specific genes, e.g. Egr2, upregulates myelin-related transcripts, it remains unknown if such manipulation can functionalize the cells and enhance their myelination frequency. The ability to do so could have implications in the use of human stem cell-derived Schwann cells, where myelination is hard to achieve. After screening four candidate transcription factors (Sox10, Oct6, Brn2 and Egr2), we found that overexpression of Egr2 in rat Schwann cells co-cultured with sensory neurons enhanced myelination frequency and reduced cell proliferation. However, in a mouse model of sciatic nerve repair with cells engrafted within a nerve guide, myelination frequency in the engrafted cells was reduced upon Egr2 overexpression. Our results show that while overexpression of Egr2 can enhance the myelination frequency in vitro, it is context-dependent, potentially influenced by the microenvironment, timing of association with axons, expression level, species differences, or other factors.

Alzheimer's disease (AD) is a progressive neurodegenerative disease currently lacking effective treatment. Efficient delivery of siRNA via nanoparticles may emerge as a viable therapeutic approach to treat AD and other central nervous system disorders. We report here the use of a linear polyethyleneimine (LPEI)-g-polyethylene glycol (PEG) copolymer-based micellar nanoparticle system to deliver siRNA targeting BACE1 and APP, two therapeutic targets of AD. Using LPEI-siRNA nanoparticles against either BACE1 or APP in cultured mouse neuroblastoma (N2a) cells, we observe selective knockdown, respectively, of BACE1 or APP. The encapsulation of siRNA by LPEI-g-PEG carriers, with different grafting degrees of PEG, leads to the formation of micellar nanoparticles with distinct morphologies, including worm-like, rod-like, or spherical nanoparticles. By infusing these shaped nanoparticles into mouse lateral ventricles, we show that rod-shaped nanoparticles achieved the most efficient knockdown of BACE1 in the brain. Furthermore, such knockdown is evident in spinal cords of these treated mice. Taken together, our findings indicate that the shape of siRNA-encapsulated nanoparticles is an important determinant for their delivery and gene knockdown efficiency in the central nervous system.

In microvascular vessels, endothelial cells are aligned longitudinally whereas several components of the extracellular matrix (ECM) are organized circumferentially. While current three-dimensional (3D) in vitro models for microvasculature have allowed the study of ECM-regulated tubulogenesis, they have limited control over topographical cues presented by the ECM and impart a barrier for the high-resolution and dynamic study of multicellular and extracellular organization. Here we exploit a 3D fibrin microfiber scaffold to develop a novel in vitro model of the microvasculature that recapitulates endothelial alignment and ECM deposition in a setting that also allows the sequential co-culture of mural cells. We show that the microfibers' nanotopography induces longitudinal adhesion and alignment of endothelial colony-forming cells (ECFCs), and that these deposit circumferentially organized ECM. We found that ECM wrapping on the microfibers is independent of ECFCs' actin and microtubule organization, but it is dependent on the curvature of the microfiber. Microfibers with smaller diameters (100-400 µm) guided circumferential ECM deposition, whereas microfibers with larger diameters (450 µm) failed to support wrapping ECM. Finally, we demonstrate that vascular smooth muscle cells attached on ECFC-seeded microfibers, depositing collagen I and elastin. Collectively, we establish a novel in vitro model for the sequential control and study of microvasculature development and reveal the unprecedented role of the endothelium in organized ECM deposition regulated by the microfiber curvature.

Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis.The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34(+) cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34(+) cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34(+) cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34(+) cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality.These findings demonstrate a novel approach utilizing nanofiber-expanded CD34(+) cells as a therapeutic application for the treatment of osteoporosis.

Therapeutic potential was evaluated in a rat model of myocardial infarction using nanofiber-expanded human cord blood derived hematopoietic stem cells (CD133+/CD34+) genetically modified with VEGF plus PDGF genes (VIP).Myocardial function was monitored every two weeks up to six weeks after therapy. Echocardiography revealed time dependent improvement of left ventricular function evaluated by M-mode, fractional shortening, anterior wall tissue velocity, wall motion score index, strain and strain rate in animals treated with VEGF plus PDGF overexpressed stem cells (VIP) compared to nanofiber expanded cells (Exp), freshly isolated cells (FCB) or media control (Media). Improvement observed was as follows: VIP>Exp> FCB>media. Similar trend was noticed in the exercise capacity of rats on a treadmill. These findings correlated with significantly increased neovascularization in ischemic tissue and markedly reduced infarct area in animals in the VIP group. Stem cells in addition to their usual homing sites such as lung, spleen, bone marrow and liver, also migrated to sites of myocardial ischemia. The improvement of cardiac function correlated with expression of heart tissue connexin 43, a gap junctional protein, and heart tissue angiogenesis related protein molecules like VEGF, pNOS3, NOS2 and GSK3. There was no evidence of upregulation in the molecules of oncogenic potential in genetically modified or other stem cell therapy groups.Regenerative therapy using nanofiber-expanded hematopoietic stem cells with overexpression of VEGF and PDGF has a favorable impact on the improvement of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction.