Xiao-Bing Zhang

Efficient Reprogramming of Human Cord Blood CD34+ Cells Into Induced Pluripotent Stem Cells With OCT4 and SOX2 Alone

The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34+ cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34+ cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood Using BCL-XL Together with Yamanaka Factors

The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source—peripheral blood—has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34+ cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3−/CD19− cells or T/B cell-depleted MNCs for 4–6 days led to the generation of 20–30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Rapid and efficient reprogramming of human fetal and adult blood CD34+ cells into mesenchymal stem cells with a single factor.

The direct conversion of skin cells into somatic stem cells has opened new therapeutic possibilities in regenerative medicine. Here, we show that human induced mesenchymal stem cells (iMSCs) can be efficiently generated from cord blood (CB)- or adult peripheral blood (PB)-CD34+ cells by direct reprogramming with a single factor, OCT4. In the presence of a GSK3 inhibitor, 16% of the OCT4-transduced CD34+ cells are converted into iMSCs within 2 weeks. Efficient direct reprogramming is achieved with both episomal vector-mediated transient OCT4 expression and lentiviral vector-mediated OCT4 transduction. The iMSCs express MSC markers, resemble bone marrow (BM)-MSCs in morphology, and possess in vitro multilineage differentiation capacity, yet have a greater proliferative capacity compared with BM-MSCs. Similar to BM-MSCs, the implanted iMSCs form bone and connective tissues, and are non-tumorigenic in mice. However, BM-MSCs do not, whereas iMSCs do form muscle fibers, indicating a potential functional advantage of iMSCs. In addition, we observed that a high level of OCT4 expression is required for the initial reprogramming and the optimal iMSC self-renewal, while a reduction of OCT4 expression is required for multilineage differentiation. Our method will contribute to the generation of patient-specific iMSCs, which could have applications in regenerative medicine. This discovery may also facilitate the development of strategies for direct conversion of blood cells into other types of cells of clinical importance.

Cellular and molecular mechanisms of accelerated fracture healing by COX2 gene therapy: Studies in a mouse model of multiple fractures

This study sought to determine the cellular and molecular mechanisms of cyclooxygenase-2 (COX2) gene therapy to accelerate fracture repair in a mouse multiple tibial fractures model. The lenti-COX2 (or lenti-gfp control vector) was injected into fractures on day 1 post-fracture. At days 3-7, the COX2 treatment increased Sdf1-, Cxcr4-, Nes-, and Podxl-expressing mesenchymal stem cells (MSCs) within fracture calluses, suggesting an enhanced MSC recruitment or expansion. The COX2-treated mice formed smaller cartilaginous calluses that had less cartilage tissues than control mice. The expression of Sox9 mRNA was 7-fold less in COX2-treated than in control calluses at day 14, implying that COX2 reduces chondrocytic differentiation of MSCs. The therapy also enhanced angiogenesis as reflected by increased immunostaining of CD31, vWF, and α-SMA over controls in the cartilaginous callus at day 14-21. At which time, the COX2 gene therapy promoted bony remodeling of the cartilaginous callus to bridge the fracture gap that was accompanied by 2-fold increase in osteoclasts along the surface of the woven bone and an onset of osteogenesis. Blocking angiogenesis with daily injection of endostatin from day 4 to day 10 into fracture sites blocked the COX2-mediated reduction of callus size that was associated with an increase in hypertrophic chondrocytes and concomitant reduction in osteoclasts. In conclusion, COX2 accelerates fracture healing in part through three biological actions: 1) increased recruitment/expansion of MSCs; 2) decreased cartilaginous callus formation; and 3) increased angiogenesis-dependent cartilage remodeling. These effects were associated with an earlier onset of bony bridging of the fracture gap.

Erythroid Promoter Confines FGF2 Expression to the Marrow after Hematopoietic Stem Cell Gene Therapy and Leads to Enhanced Endosteal Bone Formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1+ hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1+ cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation.

PDGFB-based stem cell gene therapy increases bone strength in the mouse

Significance Osteoporosis is a morbid disease afflicting millions of people worldwide. To unlock the unique regenerative powers of the skeleton that have not yet been exploited, we used stem cell gene therapy to dramatically increase bone formation at sites where bone is lost during osteoporosis. Our therapy tremendously increased de novo trabecular bone formation and trabecular connections, resulting in a large increase in bone strength. Our therapy has clinical potential, may serve as a prototype for future skeletal stem cell gene therapies, and is a model for mechanistic studies of de novo trabecular bone formation. Substantial advances have been made in the past two decades in the management of osteoporosis. However, none of the current medications can eliminate the risk of fracture and rejuvenate the skeleton. To this end, we recently reported that transplantation of hematopoietic stem/progenitor cells (HSCs) or Sca1+ cells engineered to overexpress FGF2 results in a significant increase in lamellar bone matrix formation at the endosteum; but this increase was attended by the development of secondary hyperparathyroidism and severe osteomalacia. Here we switch the therapeutic gene to PDGFB, another potent mitogen for mesenchymal stem cells (MSCs) but potentially safer than FGF2. We found that modest overexpression of PDGFB using a relatively weak phosphoglycerate kinase (PGK) promoter completely avoided osteomalacia and secondary hyperparathyroidism, and simultaneously increased trabecular bone formation and trabecular connectivity, and decreased cortical porosity. These effects led to a 45% increase in the bone strength. Transplantation of PGK-PDGFB–transduced Sca1+ cells increased MSC proliferation, raising the possibility that PDGF-BB enhances expansion of MSC in the vicinity of the hematopoietic niche where the osteogenic milieu propels the differentiation of MSCs toward an osteogenic destination. Our therapy should have potential clinical applications for patients undergoing HSC transplantation, who are at high risk for osteoporosis and bone fractures after total body irradiation preconditioning. It could eventually have wider application once the therapy can be applied without the preconditioning.

Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Peripheral blood is the easy-to-access, minimally invasive, and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol, one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood.

Few Single Nucleotide Variations in Exomes of Human Cord Blood Induced Pluripotent Stem Cells

The effect of the cellular reprogramming process per se on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs) reprogrammed from human cord blood (CB) CD34+ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2–14%) reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS), OS and ZSCAN4 (OSZ), OS and MYC and KLF4 (OSMK). Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs), in exomes, including untranslated regions (UTR), in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.

Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency

CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60–70%) and MSCs (65–75%). Furthermore, we observed a decrease of 10–20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable.

A novel xenograft model to study the role of TSLP-induced CRLF2 signals in normal and malignant human B lymphopoiesis

Thymic stromal lymphopoietin (TSLP) stimulates in vitro proliferation of human fetal B-cell precursors. However, its in vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (−T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in −T mice. Patient-derived xenografts generated from +T as compared to −T mice showed a 3–6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from −T mice. +T/−T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.