Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.

https://hsci.harvard.edu/files/hsci/files/pagliuca_et_al_cell_2014.pdf

Generation of functional human pancreatic β cells in vitro

Remyelination involves reinvesting demyelinated axons with new myelin sheaths. In stark contrast to the situation that follows loss of neurons or axonal damage, remyelination in the CNS can be a highly effective regenerative process. It is mediated by a population of precursor cells called oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, despite its efficiency in experimental models and in some clinical diseases, remyelination is often inadequate in demyelinating diseases such as multiple sclerosis (MS), the most common demyelinating disease and a cause of neurological disability in young adults. The failure of remyelination has profound consequences for the health of axons, the progressive and irreversible loss of which accounts for the progressive nature of these diseases. The mechanisms of remyelination therefore provide critical clues for regeneration biologists that help them to determine why remyelination fails in MS and in other demyelinating diseases and how it might be enhanced therapeutically.

Remyelination in the CNS: from biology to therapy

Demyelination contributes to loss of function after spinal cord injury, and thus a potential therapeutic strategy involves replacing myelin-forming cells. Here, we show that transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into adult rat spinal cord injuries enhances remyelination and promotes improvement of motor function. OPCs were injected 7 d or 10 months after injury. In both cases, transplanted cells survived, redistributed over short distances, and differentiated into oligodendrocytes. Animals that received OPCs 7 d after injury exhibited enhanced remyelination and substantially improved locomotor ability. In contrast, when OPCs were transplanted 10 months after injury, there was no enhanced remyelination or locomotor recovery. These studies document the feasibility of predifferentiating hESCs into functional OPCs and demonstrate their therapeutic potential at early time points after spinal cord injury.

https://www.jneurosci.org/content/jneuro/25/19/4694.full.pdf

Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.

Genetically engineered mice are used extensively to examine molecular responses to spinal cord injury (SCI). Inherent strain differences may confound behavioral outcomes; therefore, behavioral characterization of several strains after SCI is warranted. The Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) for rats has been widely used for SCI mice, but may not accurately reflect their unique recovery pattern. This study's purpose was to develop a valid locomotor rating scale for mice and to identify strain differences in locomotor recovery after SCI. We examined C57BL/6, C57BL/10, B10.PL, BALB/c, and C57BL/6x129S6 F1 strains for 42 days after mild, moderate, and severe contusive SCI or transection of the mid thoracic spinal cord. Contusions were created using the Ohio State University electromagnetic SCI device which is a displacement-driven model, and the Infinite Horizon device, which is a force-driven model. Attributes and rankings for the Basso Mouse Scale for Locomotion (BMS) were determined from frequency analyses of seven locomotor categories. Mouse recovery differed from rats for coordination, paw position and trunk instability. Disagreement occurred across six expert raters using BBB (p < 0.05) but not BMS to assess the same mice. BMS detected significant differences in locomotor outcomes between severe contusion and transection (p < 0.05) and SCI severity gradations resulting from displacement variations of only 0.1 mm (p < 0.05). BMS demonstrated significant face, predictive and concurrent validity. Novice BMS raters with training scored within 0.5 points of experts and demonstrated high reliability (0.92-0.99). The BMS is a sensitive, valid and reliable locomotor measure in SCI mice. BMS revealed significantly higher recovery in C57BL/10, B10.PL and F1 than the C57BL/6 and BALB/c strains after moderate SCI (p < 0.05). The differing behavioral response to SCI suggests inherent genetic factors significantly impact locomotor recovery and must be considered in studies with inbred or genetically engineered mouse strains.

Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.

L'invention concerne des populations de cellules neuronales portant des marqueurs de cellules gliales, p. ex. oligodendrocytes et leurs precurseurs. Les populations sont produites par la differenciation de cellules souches myeloides telles que des cellules souches embryonnaires humaines, dans des conditions qui favorisent l'enrichissement des cellules par un phenotype ou une capacite fonctionnelle voulus. Diverses combinaisons de facteurs de differenciation et de mitogenes peuvent etre utilisees pour produire des populations de cellules portant des marqueurs de cellules precurseurs d'oligodendrocytes. Une differenciation ulterieure produit des processus complexes caracteristiques d'oligodendrocytes a maturite. Les cellules peuvent former des gaines de myeline et peuvent etre utilisees a des fins therapeutiques pour ameliorer la fonction du systeme nerveux central.

Oligodendrocytes derives de cellules souches embryonnaires humaines pour remyelinisation et traitement de lesion de la moelle epiniere

2047 Background: Standard GBM therapy is associated with early progression and poor overall survival (OS). DC-ATA AV-GBM-1, a personal vaccine consisting of autologous DC pulsed with ATA, was investigated in a multicenter trial in patients with newly diagnosed GBM. Methods: Key eligibility criteria for surgical collection of tumor were clinical suspicion of new primary GBM & age 18-70 years. ATA lysate was prepared from irradiated tumor cells that were self-renewing in serum-free media. Autologous monocytes (MC) were collected by leukapheresis. Prior to initiating concurrent radiation therapy (RT) and temozolomide (TMZ), patients were enrolled with intent-to-treat (ITT) with DC-ATA after RT/TMZ. Eligibility included confirmation of primary GBM, availability of ATA & MC, KPS > 70 and plans for RT/TMZ. DC-ATA was manufactured during RT/TMZ. MC were differentiated into DC by culturing with IL-4 & GM-CSF, then DC were incubated with ATA. DC-ATA was suspended in 500 mg GM-CSF just prior to s.c. injections at weeks 1, 2, 3, 8, 12,16, 20, & 24 (8 doses). Patients were not excluded based on apparent disease progression or PFS. Standard adjuvant TMZ regimens were started after the 3 weekly injections. Primary endpoint was > 75% OS 14.6 months from ITT enrollment. Secondary endpoints included median OS & progression-free survival (PFS). Results: Cell line and MC collection were successful for 97% of patients. Median age of the 60 ITT enrollees was 59 years. 3 patients withdrew before starting DC-ATA; 57 received 392 injections; 68% received all 8. Most common AE attributed to DC-ATA were local injection site reactions (16%) & flu-like symptoms (10%), but 33% experienced seizures. After 3 years of follow up, OS at 14.6 mos is 52.7% (95% CI 39.8,65.8), median OS 16.0 mos (95% CI 12.9,21.7) & median PFS 10.4 mos (95% CI 8.6,11.6). OS rates at 1, 2, & 3 years are 70.1%, 32.4%, & 23.2%. Longer OS was associated with 8 DC-ATA doses (p < 0.0001), on < 2 mg/day dexamethasone (dex) at start of DC-ATA (p = 0.005), > 6 cycles of adjuvant TMZ (p = 0.0054), & KPS 90 or 100 (p = 0.010) at enrollment. Independent variables per multivariate Cox regression analysis were 8 DC-ATA doses, dex dose, IDH mutated, TMZ > 6 cycles, & MGMT promoter methylated. Concurrent TMZ regimens included TMZ alone (n = 28), TMZ + anti-VEGF(n = 14), & TMZ + tumor treating fields (TTF) (n = 10); 8 received no concurrent TMZ. OS was longer in patients treated with concurrent TMZ alone compared to no TMZ (p = 0.0003), TMZ + anti-VEGF (p = 0.045) or TMZ + TTF (p = 0.045). The only common features among 7 patients progression-free at 3 years are 8 DC-ATA injections, age < 60, & < 2 mg dex. Conclusions: DC-ATA was reliably produced and injections well-tolerated in combination with various TMZ-based regimens, but the primary OS objective was not achieved. PFS was encouraging, but did not translate into improved OS, perhaps because DC-ATA was limited to 8 injections. Clinical trial information: NCT03400917 .

Final results of phase 2 trial of personal dendritic cell (DC) vaccines loaded with autologous tumor antigens (ATA) in newly diagnosed glioblastoma (GBM).

<h3>Background</h3> Despite standard therapy, which includes chemotherapy and aggressive surgical debulking, relative 5-year survival for newly diagnosed advanced epithelial ovarian cancer is only 30%. AV-OVA-1 is a personal vaccine consisting of autologous dendritic cells (DC) loaded ex vivo with autologous tumor antigens (ATA) from a lysate of irradiated tumor cells from short-term cell culture. After completion of primary chemotherapy, immunization with DC-ATA may improve survival. A multi-center, 2:1 double-blind randomized phase 2 clinical trial was designed to compare treatment with AV-OVA-1 to autologous monocytes (MC). One objective was to determine the feasibility of collecting sufficient MC from which to generate DC for pulsing with ATA from ovarian cancer tumor-initiating cells (TIC) to produce DC-ATA. <h3>Methods</h3> Peripheral blood mononuclear cells (PBMC) were collected by leukapheresis per local standard operating procedures, then shipped by overnight courier to the AIVITA laboratory in Irvine, CA. The product was enriched for MC using the Elutra® Cell Separation System (Terumo, Lakewood, CO.). If fewer than 450 million viable MC were available for cryopreservation, an additional leukapheresis was allowed. MC were cryopreserved in liquid nitrogen and subsequently thawed and incubated in media containing granulocyte-macrophage colony-stimulating factor and interleukin-4 to differentiate MC into DC. Batches of patient-specific AV-GBM-1 were produced by incubating autologous DC with a lysate of irradiated TICs, then aliquoted into individual doses. <h3>Results</h3> Patients were enrolled from five sites in California and one in Colorado. PBMC were collected between December 2017 and April 2021. 47/50 patients (94%) had a successful PBMC collection, but four required a second procedure. An average of 1.90 billion monocytes (range 0.527 to 16.6 billion) were collected with an average of 1.55 billion monocytes cryopreserved (range 0.249 to 3.80), which subsequently could be thawed and differentiated into DC. For 29 patients treated with DC-ATA, an average of 648 million viable MC (range 336 to 1120) were differentiated into an average of 410 million DC (range 171 to 892) for incubation with ATA, which yielded an average of 78.6 million DC-ATA (range 16.0 to 270). This resulted in an average of 8.1 million DC-ATA cells per dose (range 2.0 to 27). <h3>Conclusions</h3> Leukapheresis procedures reliably resulted in collection of sufficient numbers of monocytes to generate DC and large batches of personal AV-OVA-1 vaccines. <h3>Trial Registration</h3> Clinicaltrials. gov NCT00331526 <h3>Ethics Approval</h3> This study was approved by the Western IRB, approval number 20171661; all participants gave written informed consent before taking part 

/pdf/565-leukapheresis-to-obtain-monocytes-to-produce-dendritic-3ex3f4vo.pdf

565 Leukapheresis to obtain monocytes to produce dendritic cells for manufacturing AV-OVA-1 personal vaccines in a randomized phase II trial in patients with newly diagnosed advanced ovarian cancer


 Standard aggressive therapy for primary newly diagnosed glioblastoma (GBM), which includes surgical resection followed by concurrent radiation therapy and temozolomide chemotherapy (RT/TMZ) and then maintenance TMZ, is associated with poor survival rates. Adding treatment with AV-GBM-1, a personal vaccine consisting of autologous dendritic cells (DC) pulsed with autologous tumor antigens (ATA) may improve survival. AV-GBM-1 is produced by incubating ATA with autologous DC. ATA are from a lysate of irradiated autologous GBM cells that were self-renewing in serum-free medium with factors that favor survival and proliferation of tumor initiating cells, i.e., stem cells and early progenitor cells. After recovery from RT/TMZ, GBM patients were injected subcutaneously with AV-GBM-1 admixed in granulocyte-macrophage colony-stimulating factor (GM-CSF) at weeks 1, 2, 3, 8, 12, 16, 20, and 24. This study examined changes in plasma proteomics before and after injections of AV-GBM-1. 57 patients were treated during November 2018 to October 2020. Median progression-free and overall survival were 10.3 and 16.0 months respectively. Plasma samples obtained at baseline (week-0), and just prior to the third injection (week-2) were cryopreserved and subsequently analyzed for 448 proteomic markers using quantitative, multiplex enzyme-linked immunosorbent assays (Raybiotech, Inc., Norcross, GA.). 54 patients had a baseline week-0 sample prior to the 1st vaccination; 50 had a week-2 sample just prior to the 3rd dose; 49 had paired samples for both time-points. The averages of paired samples were measured for all patients and for cohorts defined by median survival that were compared by 2-tail T-Tests. After two weekly injections there were significant (p<0.01) increases in thymus and activation-regulated chemokine (TARC, CCL17), chemerin, lipocalin-2 and angiopoietin, and decreases in thrombospondin-5, angiotensinogen, and beta-fibroblast growth factor. The changes in TARC are due to GM-CSF. 25 patients survived more than 15 months and 24 less than 15 months. Prognostic baseline markers associated with longer survival were higher CD14 (p=0.009), higher tissue-inhibitor of metalloproteinases (TIMP-1), and higher insulin growth factor binding proteins (IGFBP) 1 and 2 (p<0.02). The only markers that appeared predictive for survival (p<0.05) based on changes from baseline to week-2 were increased lipocalin-2, associated with longer survival (p=0.0369), and decreased brain-derived neurotrophic factor (BDNF) associated with shorter survival (p=0.0186). Because of the large number of analyses, these results are considered hypothesis generating and not definitive.
 Citation Format: Daniela A. Bota, David E. Piccioni, Christopher M. Duma, Renato V. LaRoca, Santosh Kesari, Mehrdad Abedi, Robert D. Aiken, Aleksandra J. Poole, Gabriel I. Nistor, Robert O. Dillman. Plasma proteomic markers prognostic or predictive for survival of patients with newly diagnosed glioblastoma who were treated in a phase II trial with standard care and the addition of the novel patient-specific dendritic cell vaccine AV-GBM-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT571.

Abstract CT571: Plasma proteomic markers prognostic or predictive for survival of patients with newly diagnosed glioblastoma who were treated in a phase II trial with standard care and the addition of the novel patient-specific dendritic cell vaccine AV-GBM-1

Gabriel Nistor

Papers

Oligodendrocytes derives de cellules souches embryonnaires humaines pour remyelinisation et traitement de lesion de la moelle epiniere

Final results of phase 2 trial of personal dendritic cell (DC) vaccines loaded with autologous tumor antigens (ATA) in newly diagnosed glioblastoma (GBM).

565 Leukapheresis to obtain monocytes to produce dendritic cells for manufacturing AV-OVA-1 personal vaccines in a randomized phase II trial in patients with newly diagnosed advanced ovarian cancer

Abstract CT571: Plasma proteomic markers prognostic or predictive for survival of patients with newly diagnosed glioblastoma who were treated in a phase II trial with standard care and the addition of the novel patient-specific dendritic cell vaccine AV-GBM-1