Wei Hong Chen

Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging

The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with “tracer” hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK+, EGFP+ stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with 18F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([18F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK+ tumor stroma with an average of 0.36 ± 0.24% ID/g [18F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF+ and CD31+ endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK+, GFP+ endothelial cells had limited proliferative capacity and a short life span of <2 weeks in tumor fragments transplanted into secondary hosts. We conclude that hMSCs can target microscopic tumors, subsequently proliferate and differentiate, and contribute to formation of a significant portion of tumor stroma. PET imaging should facilitate clinical translation of stem cell–based anticancer gene therapeutic approaches by providing the means for in vivo noninvasive whole-body monitoring of trafficking, tumor targeting, and proliferation of HSV1-tk-expressing “tracer” hMSCs in tumor stroma.

In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

OBJECTIVE Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)-based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration. METHODS A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage-forming ability. RESULTS Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold. CONCLUSION These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell-based transplantation for future clinical use.

Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-β1 in platelet-rich plasma

Human intervertebral disc (IVD) degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor‐β1 (TGF‐β1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In this study, we examine the role of TGF‐β1 in platelet‐rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue‐engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was 1 ng/ml TGF‐β1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time‐dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT‐PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p‐Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue‐engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue‐engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration. J. Cell. Physiol. 209: 744–754, 2006.

Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma

An ex vivo degenerative intervertebral disc (IVD) organ culture system was established for the screening of disc regeneration agents. Its application was demonstrated by a stem cell and growth factor-based therapeutic approach for the amelioration of IVD. An ex vivo culture system using chymopapain to partially digest nucleus proposus tissue was established to mimic human IVD degeneration. This system was then used for the evaluation of different therapeutic regimens including: mesenchymal stem cell derived from eGFP-transgenic porcine (MSC-GFP), platelet-rich plasma (PRP) and MSC-GFP/PRP combined treatment, and confirmed in in vivo animal model. Chondrogenic-specific gene products including Col II and aggrecan were found upregulated and chondrogenic matrix deposition increased, as evident by sustained fluorescent signals over 4 weeks, in the MSC-GFP implanted group. Previously, we demonstrated in vitro stage-specific chondrogenesis of MSC by chondrocytic commitment. These same molecules upregulated for chondrogenesis were also observed in MSC-GFP group. PRP that has been shown to promote nucleus pulposus (NP) regeneration also resulted in significant increased levels of mRNA involved in chondrogenesis and matrices accumulation. The ex vivo IVD regeneration results were repeated and supported by in vivo porcine degenerative system. Moreover, the disc height index (DHI) was significantly increased in both in vivo MSC-GFP and PRP regeneration groups. Unexpectedly, the MSC-GFP/PRP combined therapy demonstrated an inclination towards osteogenesis in ex vivo system. The ex vivo degenerative IVD culture system described in this study could serve as an alternative and more accessible model over large animal model. This system also provides a high-throughput platform for screening therapeutic agents for IVD regeneration.

Regenerative potentials of platelet-rich plasma enhanced by collagen in retrieving pro-inflammatory cytokine-inhibited chondrogenesis

This study was undertaken to evaluate the role of collagen matrix to enhance platelet-rich plasma (PRP) effects on pro-inflammatory cytokine-induced arthritic model. We have previously demonstrated the highly regenerative roles of PRP to restore disc degeneration and osteoporosis. In this study, PRP modulated by collagen matrix was used as a regenerative and anti-inflammatory mediator to rescue the chondrocyte degeneration induced by pro-inflammatory cytokines IL-1β (10 ng/ml)+TNF-α (20 ng/ml). First, the MTT result indicated that 1 ng/ml TGF-β1 in PRP showed an optimal dosage for chondrocytes proliferation. The chondrogenic-specific gene expressions were rescued by PRP from the inhibition of IL-1β+TNF-α, especially under the modulation of collagen matrix. The inflammatory molecules activated by IL-1β+TNF-α were also significantly diminished by PRP with collagen matrix. The membrane receptors integrin α1β1 and CD44 were strongly inhibited by IL-1β+TNF-α, while this inhibition was then recovered by PRP in collagen coating condition. In a 3D model encapsulated with collagen, PRP-induced chondrogenesis were highly enhanced, such as strong restoration of type II collagen and proteoglycan from the inhibition of IL-1β+TNF-α. The result indicated that collagen matrix enhances the effect of PRP on chondrogenesis in response to pro-inflammatory cytokines. The combination of PRP and collagen matrix might facilitate a physiological microenvironment beneficial for maintaining chondrocyte homeostasis and represents an advanced osteoarthritis therapy for clinical applications.

Synergistic anabolic actions of hyaluronic acid and platelet-rich plasma on cartilage regeneration in osteoarthritis therapy

Osteoarthritis (OA) is a common disease associated with tissue inflammation, physical disability and imbalanced homeostasis in cartilage. For advanced treatments, biological approaches are currently focused on tissue regeneration and anti-inflammation. This study was undertaken to evaluate the therapeutic efficacies of hyaluronic acid (HA) and platelet-rich plasma (PRP) (HA+PRP) on OA. Articular chondrocytes were obtained from five OA patients. The optimal HA and PRP concentrations were evaluated by MTT assay. The expressions of chondrogenic and inflammatory genes were analyzed by RT-PCR. Signaling pathway was examined by immunoblotting and the expressions of OA pathology-related chemokines and cytokines was demonstrated by real-time PCR-based SuperArray. The therapeutic efficacies of HA+PRP were then demonstrated in 3D arthritic neo-cartilage and ACLT-OA model. Here we showed that HA+PRP could greatly retrieve pro-inflammatory cytokines-reduced articular chondrocytes proliferation and chondrogenic phenotypes, the mechanism of which involve the sequential activation of specific receptors CD44 and TGF-βRII, downstream mediators Smad2/3 and Erk1/2, and the chondrogenic transcription factor SOX9. The real-time PCR-based SuperArray results also indicated that OA pathology-related chemokines and cytokines could be efficiently suppressed by HA+PRP. Moreover, the cartilaginous ECM could be retrieved from inflammation-induced degradation by HA+PRP in both 2D monolayer and 3D neo-cartilage model. Finally, the intra-articular injection of HA+PRP could strongly rescue the meniscus tear and cartilage breakdown and then decrease OA-related immune cells. The combination of HA+PRP can synergistically promote cartilage regeneration and inhibit OA inflammation. This study might offer an advanced and alternative OA treatment based on detailed regenerative mechanisms.

Potential Osteoporosis Recovery by Deep Sea Water through Bone Regeneration in SAMP8 Mice

The aim of this study is to examine the therapeutic potential of deep sea water (DSW) on osteoporosis. Previously, we have established the ovariectomized senescence-accelerated mice (OVX-SAMP8) and demonstrated strong recovery of osteoporosis by stem cell and platelet-rich plasma (PRP). Deep sea water at hardness (HD) 1000 showed significant increase in proliferation of osteoblastic cell (MC3T3) by MTT assay. For in vivo animal study, bone mineral density (BMD) was strongly enhanced followed by the significantly increased trabecular numbers through micro-CT examination after a 4-month deep sea water treatment, and biochemistry analysis showed that serum alkaline phosphatase (ALP) activity was decreased. For stage-specific osteogenesis, bone marrow-derived stromal cells (BMSCs) were harvested and examined. Deep sea water-treated BMSCs showed stronger osteogenic differentiation such as BMP2, RUNX2, OPN, and OCN, and enhanced colony forming abilities, compared to the control group. Interestingly, most untreated OVX-SAMP8 mice died around 10 months; however, approximately 57% of DSW-treated groups lived up to 16.6 months, a life expectancy similar to the previously reported life expectancy for SAMR1 24 months. The results demonstrated the regenerative potentials of deep sea water on osteogenesis, showing that deep sea water could potentially be applied in osteoporosis therapy as a complementary and alternative medicine (CAM).

Preferential therapy for osteoarthritis by cord blood MSCs through regulation of chondrogenic cytokines

Osteoarthritis (OA) is a common rheumatic disease associated with imbalanced cartilage homeostasis which could be corrected by mesenchymal stem cells (MSCs) therapy. However, MSCs from different origins might exhibit distinct differentiation capacities. This study was undertaken to compare the therapeutic efficacies between MSCs from cord blood (CB-MSCs) and bone marrow (BM-MSCs) on OA treatment. The surface phenotypes and multipotent capacities of CB-MSCs and BM-MSCs were first characterized. The coculture commitment system was subsequently utilized for comparing the patterned molecules in stage-specific chondrogenesis of committed MSCs. For examining the therapeutic efficacies, committed CB-MSCs and BM-MSCs were encapsulated in neo-cartilage and subjected into pro-inflammatory cytokine environment. Finally, chondrogenic and inflammatory cytokine profiles in committed MSCs were evaluated. CB-MSCs and BM-MSCs were both negative for hematopoietic markers and positive for adhesion and mesenchymal cell markers. The CB-MSCs showed a markedly higher chondrogenic potential and relatively lower osteogenic and adipogenic capacities than BM-MSCs. During chondrogenesis, the committed CB-MSCs also showed significant increases in cell proliferation, adhesion molecules, signaling molecules, and chondrogenic-specific gene expressions in a coculture system. For the therapeutic efficacies, the committed CB-MSCs could strongly recover the pro-inflammatory cytokines diminished-Col II and proteoglycan expressions in a 3D arthritic model. The IL-10, ICAM-1 and TGF-β1 were also up-regulated in committed CB-MSCs analyzed by using cytokine profiling. Our data demonstrate that CB-MSCs possess specific advantages in cartilage regeneration over BM-MSCs. The CB-MSCs showed a better therapeutic potential that can contribute to advanced cell-based transplantation for clinical OA therapy.

A novel cell-based therapy for contusion spinal cord injury using GDNF-delivering NIH3T3 cells with dual reporter genes monitored by molecular imaging

This aim of our study was to evaluate a novel cell-based therapy for contusion spinal cord injury (SCI) using embryonic-derived NIH3T3 cells, which endogenously express glial cell line–derived neurotrophic factor (GDNF). Methods: Proliferation and differentiation of transplanted NIH3T3 cells and their anti-apoptotic effects were examined after their engraftment into the spinal cords of Long-Evans rats subjected to acute SCI at the T10 vertebral level by a New York University impactor device. NIH3T3 cells were initially engineered to contain dual reporter genes, namely thymidine kinase (T) and enhanced green fluorescence protein (G), for in vivo cell tracking by both nuclear and fluorescence imaging modalities. Results: Planar and fluorescence imaging demonstrated that transplanted NIH3T3-TG cells at the L1 vertebral level migrated 2 cm distal to the injury site as early as 2 h, and the signals persisted for 48 h after SCI. The expression of GDNF by NIH3T3-TG cells was then confirmed by immunohistochemical analysis both in vitro and in vivo. GDNF-secreting NIH3T3-TG transplant provided anti-apoptotic effects in the injured cord over the period of 3 wk. Finally, NIH3T3-TG cells cultured under neuronal differentiation medium exhibited both morphologic and genetic resemblance to neuronal cells. Conclusion: GDNF-secreting NIH3T3-TG cells in combination with molecular imaging could be a platform for developing therapeutic tools for acute SCI.

Delayed animal aging through the recovery of stem cell senescence by platelet rich plasma.

Aging is related to loss of functional stem cell accompanying loss of tissue and organ regeneration potentials. Previously, we demonstrated that the life span of ovariectomy-senescence accelerated mice (OVX-SAMP8) was significantly prolonged and similar to that of the congenic senescence-resistant strain of mice after platelet rich plasma (PRP)/embryonic fibroblast transplantation. The aim of this study is to investigate the potential of PRP for recovering cellular potential from senescence and then delaying animal aging. We first examined whether stem cells would be senescent in aged mice compared to young mice. Primary adipose derived stem cells (ADSCs) and bone marrow derived stem cells (BMSCs) were harvested from young and aged mice, and found that cell senescence was strongly correlated to animal aging. Subsequently, we demonstrated that PRP could recover cell potential from senescence, such as promote cell growth (cell proliferation and colony formation), increase osteogenesis, decrease adipogenesis, restore cell senescence related markers and resist the oxidative stress in stem cells from aged mice. The results also showed that PRP treatment in aged mice could delay mice aging as indicated by survival, body weight and aging phenotypes (behavior and gross morphology) in term of recovering the cellular potential of their stem cells compared to the results on aged control mice. In conclusion these findings showed that PRP has potential to delay aging through the recovery of stem cell senescence and could be used as an alternative medicine for tissue regeneration and future rejuvenation.