T-Cell Lymphomas in Recipients of CAR-T Cells: Assessing Risks and Causalities.

The United States Food and Drug Administration (FDA) announcement in November 2023 regarding reports of the occurrence of secondary T-cell lymphomas in patients receiving chimeric antigen receptor T-cells (CAR-T) for B-cell malignancies resulted in widespread concern among patients, clinicians and scientists. Little information relevant to assessing causality, most importantly whether CAR retroviral or lentiviral vector genomic insertions contribute to oncogenesis, was initially available. However, since that time several publications have provided clinical and molecular details on three cases showing clonal CAR vector insertions in tumor cells but without firm evidence these insertions played any role in oncogenic transformation. In addition, several other cases have been reported without vector detected in tumor cells. In addition, epidemiologic analyses as well as institutional long-term CAR-T recipient cohort studies provide important additional information suggesting the risk of T-cell lymphomas following CAR-T therapies is extremely low. This review will provide a summary of information available to date, as well as review relevant prior research suggesting a low susceptibility of mature T-cells to insertional oncogenesis and documenting the almost complete lack of T-cell transformation following natural HIV infection. Alternative factors that may predispose patients treated with CAR-T cells to secondary hematologic malignancies, including immune dysfunction and clonal hematopoiesis, are discussed and likely play a greater role than insertional mutagenesis in secondary malignancies post-CAR therapies.

Soluble APP functions as a vascular niche signal that controls adult neural stem cell number.

The molecular mechanism by which NSC number is controlled in the neurogenic regions of the adult brain is not fully understood but it has been shown that vascular niche signals regulate neural stem cell (NSC) quiescence and growth. Here, we have uncovered a role for soluble amyloid precursor protein (sAPP) as a vascular niche signal in the subventricular zone (SVZ) of the lateral ventricle of the adult mouse brain. sAPP suppresses NSC growth in culture. Further in vivo studies on the role of APP in regulating NSC number in the SVZ clearly demonstrate that endothelial deletion of App causes a significant increase in the number of BrdU label-retaining NSCs in the SVZ, whereas NSC/astrocyte deletion of App has no detectable effect on the NSC number. Taken together, these results suggest that endothelial APP functions as a vascular niche signal that negatively regulates NSC growth to control the NSC number in the SVZ.

Umbilical Cord Mesenchymal Stem Cells Derived Neurospheres Promote Long-term functional recovery But Aggravate Acute Phase Inflammation in Experimental Stroke.

Human umbilical cord mesenchymal stem cells (UC-MSCs) transplantation has been shown to ameliorate intracerebral hemorrhage (ICH) in animal and clinical studies. We previously reported an easy one-step method to induce UC-MSCs into neurospheres with much enhanced neurogenic and angiogenic potential. In the present study, we further evaluated the neuro-protective effects of these UC-MSCs derived neurospheres (UC-MSCs-NS) using a murine collagenase induced ICH model. We compared the effects of UC-MSCs or UC-MSCs-NS transplantation at two different time-points: 3 h after ICH induction (early transplantation) or three days after ICH induction (delayed transplantation). The results showed that UC-MSCs exhibited favorable effects at both time-points whereas UC-MSCs-NS early delivery led to increased cell apoptosis, exacerbated brain edema, enlarged ICH volume and deteriorated neurological function. In vivo inflammatory cytokine analysis indicated UC-MSCs transplantation was able to attenuate the acute phase secretion of inflammatory cytokines TNF-α and IL-1ß whereas UC-MSCs-NS immediate transplantation led to increased levels of these cytokines. However, long-term follow-up experiment showed delayed UC-MSCs-NS transplantation was superior to UC-MSCs transplantation alone in terms of increased neurogenic reconstitution. Our results suggest both UC-MSCs and UC-MSCs-NS can exert favorable effects in ICH therapy but the infusion of UC-MSCs-NS should avoid the super-early phase of ICH. We believe UC-MSCs derived neurospheres should be further exploited for chronic refractory neurological disorders such as chronic phase of stroke and various neurodegenerative disorders such as Alzheimer's disease.

Identification of a novel immune gene panel in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is one of the most common oral cancers. Immune activity is significantly related to the initiation and progression of TSCC. Systemic analysis of the immunogenomic landscape and identification of crucial immune-related genes (IRGs) would help understanding of TSCC. Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) provide multiple TSCC cases for use in an integrated immunogenomic study. METHODS: Immune landscape of TSCC was depicted by expression microarray data from GSE13601 and GSE34105. Univariate Cox analysis, in combination with survival analysis, was applied to select candidate IRGs with significant survival value. Survival predicting models were constructed by multivariate Cox regression and logistic regression analysis. Unsupervised clustering analysis was used to construct an immune gene panel based on prognostic IRGs to distinguish TSCC subgroups with different prognostic outcomes. Finally, IHC staining was performed to validate the clinical value of this immune-gene panel. RESULTS: Differentially expressed IRGs were identified in two TSCC microarray datasets. Functional enrichment analysis revealed that ontology terms associated with variations in T cell function, were highly enriched. Infiltration status of activated CD8+ T cells, central memory CD4+ T cells and type 17 T helper cells, had great prognostic value for TSCC progression. Unsupervised clustering analysis was further performed to classify TSCC patients into three subgroups. CTSG, CXCL13, and VEGFA were finally combined together to form an immune-gene panel, todistinguish different TSCC subgroups. IHC staining of TSCC sections further validated the clinical efficiency of the immune-gene panel consisting of prognostic IRGs to distinguish TSCC patients. CONCLUSION: VEGFA, CXCL13, and CTSG, correlated with T cell infiltration and prognostic outcome. They were screened to form an immune-gene panel to identify TSCC subgroups with different prognostic outcomes. Clinical IHC further validated the efficacy of this immune-gene panel to evaluate aggressiveness of TSCC development.

Alzheimer's Disease: From Pathogenesis to Mesenchymal Stem Cell Therapy - Bridging the Missing Link.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease worldwide. With the increasing trend of population aging, the estimated number of AD continues to climb, causing enormous medical, social and economic burden to the society. Currently, no drug is available to cure the disease or slow down its progression. There is an urgent need to improve our understanding on the pathogenesis of AD and develop novel therapy to combat it. Despite the two well-known pathological hallmarks (extracellular amyloid plaques and intracellular Neurofibrillary Tangles), the exact mechanisms for selective degeneration and loss of neurons and synapses in AD remain to be elucidated. Cumulative studies have shown neuroinflammation plays a central role in pathogenesis of AD. Neuroinflammation is actively involved both in the onset and the subsequent progression of AD. Microglia are the central player in AD neuroinflammation. In this review, we first introduced the different theories proposed for the pathogenesis of AD, focusing on neuroinflammation, especially on microglia, systemic inflammation, and peripheral and central immune system crosstalk. We explored the possible mechanisms of action of stem cell therapy, which is the only treatment modality so far that has pleiotropic effects and can target multiple mechanisms in AD. Mesenchymal stem cells are currently the most widely used stem cell type in AD clinical trials. We summarized the ongoing major mesenchymal stem cell clinical trials in AD and showed how translational stem cell therapy is bridging the gap between basic science and clinical intervention in this devastating disorder.

Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Reduced genotoxicity of avian sarcoma leukosis virus vectors in rhesus long-term repopulating cells compared to standard murine retrovirus vectors.

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Neurospheres Induced from Human Adipose-Derived Stem Cells as a New Source of Neural Progenitor Cells.

Human adipose-derived stem cells are used in regenerative medicine for treating various diseases including osteoarthritis, degenerative arthritis, cartilage or tendon injury, etc. However, their use in neurological disorders is limited, probably due to the lack of a quick and efficient induction method of transforming these cells into neural stem or progenitor cells. In this study, we reported a highly efficient and simple method to induce adipose-derived stem cells into neural progenitor cells within 12 hours, using serum-free culture combined with a well-defined induction medium (epidermal growth factor 20 ng/ml and basic fibroblast growth factor, both at 20 ng/ml, with N2 and B27 supplements). These adipose-derived stem cell-derived neural progenitor cells grow as neurospheres, can self-renew to form secondary neurospheres, and can be induced to become neurons and glial cells. Real-time polymerase chain reaction showed significantly upregulated expression of neurogenic genes Sox2 and Nestin with a moderate increase in stemness gene expression. Raybio human growth factor analysis showed a significantly upregulated expression of multiple neurogenic and angiogenic cytokines such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic growth factor, nerve growth factor, basic fibroblast growth factor and vascular endothelial growth factor etc. Therefore, adipose-derived stem cell-derived neurospheres can be a new source of neural progenitor cells and hold great potential for future cell replacement therapy for treatment of various refractory neurological diseases.

Efficient One-Step Induction of Human Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSCs) Produces MSC-Derived Neurospheres (MSC-NS) with Unique Transcriptional Profile and Enhanced Neurogenic and Angiogenic Secretomes.

Cell therapy has emerged as a promising strategy for treating neurological diseases such as stroke, spinal cord injury, and various neurodegenerative diseases, but both embryonic neural stem cells and human induced Pluripotent Stem Cell- (iPSC-) derived neural stem cells have major limitations which restrict their broad use in these diseases. We want to find a one-step induction method to transdifferentiate the more easily accessible Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSCs) into neural stem/progenitor cells suitable for cell therapy purposes. In this study, UC-MSCs were induced to form neurospheres under a serum-free suspension culture with Epidermal Growth Factor- (EGF-) and basic Fibroblast Growth Factor- (bFGF-) containing medium within 12 hours. These MSC-derived neurospheres can self-renew to form secondary neurospheres and can be readily induced to become neurons and glial cells. Real-time PCR showed significantly upregulated expression of multiple stemness and neurogenic genes after induction. RNA transcriptional profiling study showed that UC-MSC-derived neurospheres had a unique transcriptional profile of their own, with features of both UC-MSCs and neural stem cells. RayBio human growth factor cytokine array analysis showed significantly upregulated expression levels of multiple neurogenic and angiogenic growth factors, skewing toward a neural stem cell phenotype. Thus, we believe that these UC-MSC-derived neurospheres have amenable features of both MSCs and neural stem/progenitor cells and have great potential in future stem cell transplantation clinical trials targeting neurological disorders.

Transduction of rhesus macaque hematopoietic stem and progenitor cells with avian sarcoma and leukosis virus vectors.

Genome-wide integration site analyses showed that Moloney murine leukemia virus (MoMLV)- and lentivirus-derived vectors integrate preferentially into the coding regions of genes, posing a risk of insertional mutagenesis. Avian sarcoma and leukosis viruses (ASLVs) were previously reported to have a weak preference for gene-coding regions in a cell line study as compared with human immunodeficiency virus and MoMLV; however, thus far these vectors have not been studied for their potential efficacy in transduction of hematopoietic progenitor and stem cells. In this study we investigated for the first time the ability of ASLV-derived RCAS (replication-competent ALV LTR [avian leukosis virus long terminal repeat] with a splice acceptor) vectors to transduce rhesus macaque hematopoietic progenitors and long-term repopulating cells, in an autologous transplantation model. RCAS vectors can efficiently and stably transduce rhesus macaque CD34+ hematopoietic progenitor cells with an efficiency of transduction of up to 34% ex vivo. In two animals transplanted with RCAS vector-transduced autologous CD34+ cells, highly polyclonal hematopoietic reconstitution with sustained gene-marking levels in myeloid and lymphoid lineages was observed up to 18 months post-transplantation. These findings are encouraging and suggest that this vector system should be explored and further optimized for gene therapy applications targeting hematopoietic stem and progenitor cells.

Conditioned media from human adipose tissue-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells efficiently induced the apoptosis and differentiation in human glioma cell lines in vitro.

Human mesenchymal stem cells (MSCs) have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs). We found (1) MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2) MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3) real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4) furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

MicroRNA expression profiles of circulating microvesicles in hepatocellular carcinoma.

BACKGROUND: Microvesicles (MVs) packaged with microribonucleic acids (miRNAs) have been shown to be released mainly from tumor cells. However, little information is known for miRNAs from MVs in hepatocellular carcinoma (HCC). Hence, we explored the MVs' miRNAs expression profiles in HCC. METHODS: MVs were collected from peripheral blood of HCC, chronic hepatitis B (CHB) and normal controls. miRNA from MVs were labeled and analyzed by Agilent miRNA microarry. Bioinformatics tools were used to analyze function of the differentially expressed MVs' miRNAs. RESULTS: A total of 242 aberrantly expressed miRNAs were identified in HCC-MVs compared with CHB-MVs and the control. Among them, 115 miRNAs were over-expressed with up to 31 fold difference (miR-671-5p) and 127 were down-expressed with up to 0.041 fold difference (miR-432) in HCC. By software miRror2.0, nucleolar protein 3 (NOL3) was found to be the core player among 300 target genes of top ten up-regulated miRNAs and serine/ arginine repetitive matrix 1(SRRM1) was central among the 219 targets of the top ten down-regulated miRNAs. We also analyzed GO categories for these predicted genes : cellular component, biological processes, and molecular function. The deregulation of MVs' miRNAs and their target genes were closely involved in the pathways of HCC. CONCLUSIONS: Our study firstly demonstrated that miRNAs were differentially expressed in HCC-MVs compared with CHB and normal controls. Aberrant HCC-MVs miRNAs may play important roles in the development of HCC.

Side-by-side comparison of the biological characteristics of human umbilical cord and adipose tissue-derived mesenchymal stem cells.

Both human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been explored as attractive mesenchymal stem cells (MSCs) sources, but very few parallel comparative studies of these two cell types have been made. We designed a side-by-side comparative study by isolating MSCs from the adipose tissue and umbilical cords from mothers delivering full-term babies and thus compared the various biological aspects of ASCs and UC-MSCs derived from the same individual, in one study. Both types of cells expressed cell surface markers characteristic of MSCs. ASCs and UC-MSCs both could be efficiently induced into adipocytes, osteoblasts, and neuronal phenotypes. While there were no significant differences in their osteogenic differentiation, the adipogenesis of ASCs was more prominent and efficient than UC-MSCs. In the meanwhile, ASCs responded better to neuronal induction methods, exhibiting the higher differentiation rate in a relatively shorter time. In addition, UC-MSCs exhibited a more prominent secretion profile of cytokines than ASCs. These results indicate that although ASCs and UC-MSCs share considerable similarities in their immunological phenotype and pluripotentiality, certain biological differences do exist, which might have different implications for future cell-based therapy.

The molecular profiles of neural stem cell niche in the adult subventricular zone.

Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.