Bone Marrow Collection Research Articles

Mitigating oxygen stress enhances aged mouse hematopoietic stem cell numbers and function

Bone marrow (BM) hematopoietic stem cells (HSCs) become dysfunctional during aging (i.e., they are increased in number but have an overall reduction in long-term repopulation potential and increased myeloid differentiation) compared with young HSCs, suggesting limited use of old donor BM cells for hematopoietic cell transplantation (HCT). BM cells reside in an in vivo hypoxic environment yet are evaluated after collection and processing in ambient air. We detected an increase in the number of both young and aged mouse BM HSCs collected and processed in 3% O2 compared with the number of young BM HSCs collected and processed in ambient air (~21% O2). Aged BM collected and processed under hypoxic conditions demonstrated enhanced engraftment capability during competitive transplantation analysis and contained more functional HSCs as determined by limiting dilution analysis. Importantly, the myeloid-to-lymphoid differentiation ratio of aged BM collected in 3% O2 was similar to that detected in young BM collected in ambient air or hypoxic conditions, consistent with the increased number of common lymphoid progenitors following collection under hypoxia. Enhanced functional activity and differentiation of old BM collected and processed in hypoxia correlated with reduced "stress" associated with ambient air BM collection and suggests that aged BM may be better and more efficiently used for HCT if collected and processed under hypoxia so that it is never exposed to ambient air O2.

Stem cell donor registry activities during the COVID-19 pandemic: a field report by DKMS

The COVID-19 pandemic has serious implications also for patients with other diseases. Here, we describe the effects of the pandemic on unrelated hematopoietic stem cell donation and transplantation from the perspective of DKMS, a large international donor registry. Especially, we cover the development of PBSC and bone marrow collection figures, donor management including Health and Availability Check (HAC), transport and cryopreservation of stem cell products, donor recruitment and business continuity measures. The total number of stem cell products provided declined by around 15% during the crisis with a particularly strong decrease in bone marrow products. We modified donor management processes to ensure donor and product safety. HAC instead of confirmatory typing was helpful especially in countries with strict lockdowns. New transport modes were developed so that stem cell products could be safely delivered despite COVID-19-related travel restrictions. Cryopreservation of stem cell products became the new temporary standard during the pandemic to minimize risks related to transport logistics and donor availability. However, many products from unrelated donors will never be transfused. DKMS discontinued public offline donor recruitment, leading to a 40% decline in new donors during the crisis. Most DKMS employees worked from home to ensure business continuity during the crisis.

Clinical Charactistics of Ph-Negative MPN Patients with Coexistence of JAK2V617F, Calr and MPL Mutation

Background and purpose: JAK2, CALR and MPL gene mutations are the driver genes leading to the pathogenesis of ph-negative myeloproliferative meoplasm (MPN). However, it's still unkonwn about the effect of double gene mutations coexisting in ph-negative MPN patients. In this study, we analyzed the clinical significance of the three driver gene mutations when they coexisted in patients with MPN. Methods: Collected bone marrow or peripheral blood samples of 221 patients with suspected MPN who were in the Affiliated Hospital of Guizhou Medical University between May 2017 and April 2020; qPCR detection for JAK2V617F mutation and sanger sequencing detection for CALR gene exon 9 and MPL Exon 10 mutation. Searched Pubmed database related to the coexistence of the three driver gene mutations and extracted information; Fisher exact test to analyze the differences in clinical characteristics between double mutation carriers and single mutation carriers. Shapiro-Wilk statistical analysis of the differences in clinical characteristics of ET patients with different types of double mutations. Results: 1 ET and 1 PMF patient carried both JAK2V617F and CALR gene mutations were found in our center, accounting for 0.9% of ph-negative MPN. The CALR carried by PMF patients was a newly discovered mutation type, c.1126_1127insTTTGC, R376Lfs*56. Through database search, we collected 26 literatures involving MPN case reports with double-driver gene mutations. By fitting analysis with the clinical information of positive patients in our center, a total of 74 MPN patients were collected, of which the number of ET patients was the largest(57/74, accounting for 77.03%). The most common coexisting mutant type was JAK2V617F plus CALR (63/74, 85.14%). The age of onset of MPN patients with double-driver gene mutations was significantly higher than that of single-mutation carriers, and the proportion of patients over 70 years of age was higher (p<0.05), and double mutation carriers had higher platelet count levels(Plt) (p<0.05). The proportion of patients with splenomegalia was lower (p<0.05). The clinical characteristics of ET patients were similar to MPN patients, but no difference in the incidence of splenomegalia (p>0.05). Among PMF patients, the Plt and the proportion of patients with megasplenomegaly in double mutation carriers were similar to those in MPN patients, but the hemoglobin(Hb) was higher (p<0.05). In addition, we also compared the clinical characteristics of ET patients with different gene mutation types. The results showed that the Hb of patients with JAK2V617F and CALR mutations were higher than those of ET patients with JAK2V617F and MPL mutations (p<0.05), but the Plt level is low (p<0.05). Although there were no significant differences in age, gender, IPSET-thrombosis and IPSET (IWG-MRT) scores and WBC between the two groups, the proportion of female ET patients with JAKV617F with MPL mutation was significantly higher than that of JAK2V617F with CALR mutation group (p<0.05). And the proportion of patients with WBC exceeding 11×109/L was also higher (p<0.05). In addition, only one PV patient carried JAK2V617F and CALR, and achieved partial or complete remission after treatment with standard treatment regimens (hydroxyurea or interferon-α). Conclusion: JAK2V617F, CALR and MPL gene mutations are not mutually exclusive in MPN patients, but a certain number of double mutation carriers are ignored. Compared with patients with single-gene mutations, patients with double-gene mutations are older and have higher platelet counts, and should be treated differently in later treatment. At the same time, there are also differences between ET patients with different mutation combinations. These results suggest that it is very necessary for MPN patients to check for three driver gene mutations at the same time, and this type of patients may become a new subtype of MPN. Disclosures No relevant conflicts of interest to declare.

Cell-Free DNA As a Biomarker for Acute Lymphoblastic Leukemia Relapse in the Central Nervous System

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with a relapse rate of 15%. The presence of lymphoblasts in the central nervous system (CNS) is a negative prognostic indicator and a frequent site of disease relapse. CNS disease is defined as more than five white blood cells in the cerebrospinal fluid (CSF) with positive lymphoblast cytomorphology. Often leukemic blasts are detected by flow cytometry of the CSF prior to reaching the five white blood cell threshold, requiring repeat lumbar punctures at 2-4 week intervals until the arbitrarily defined clinical criteria for diagnosis are met. Additionally, more than 50% of autopsied brains from ALL patients show evidence of CNS disease, despite non-detectable CNS involvement via cytology of CSF. These data suggest that patients may be harboring CNS disease before it expands enough to be clinically diagnosed. We have developed a new assay that may allow for earlier detection of CNS disease and relapse by quantifying cell-free, circulating tumor DNA (ctDNA) in the CSF. ctDNA is a proven biomarker of relapse and metastasis in solid tumors in pre-clinical testing; however, its utility at predicting CNS disease in ALL has not been examined. We examined two possible methods for using ctDNA as a biomarker: leukemia cell clonality and DNA methylation profiling. We collected bone marrow aspirate, blood, and CSF samples from 11 newly diagnosed patients prior to the start of chemotherapy treatment to use as a training data set. ctDNA was isolated from blood and CSF samples at diagnosis and throughout treatment. Genomic DNA was isolated from bone marrow and peripheral blood mononuclear cell (PBMC) samples at diagnosis. For leukemia cell clonality assays, Invivoscribe Lymphotrack PCR assays combined with MinION (Oxford Nanopore Technologies) sequencing were used to identify the VDJ sequence of the immunoglobulin (B-ALL) or T-cell receptor (T-ALL) rearrangements in the clones comprising each leukemia. Throughout the course of treatment, ctDNA samples were run on the MinION sequencer, examining the abundance of major clones present and ctDNA quantification was done on patient plasma and CSF samples over time. While this assay relies on patient specific VDJ sequencing, we are also in the process of developing a more universal assay that utilizes recurrent methylation changes in T-ALL or B-ALL, compared to normal PBMCs. We performed methylation sequencing on 3 control PBMC samples and 7 ALL patient samples to identify differentially methylated regions (DMRs) present specifically in ALL samples. This sequencing identified 9,222 DMRs present in the ALL samples. These sites were then compared with publicly available datasets, yielding 55 overlapping regions and 19 specific overlapping methylation sites commonly present in ALL samples and not in PBMCs. We are now in the process of validating these differentially methylated sites by droplet digital polymerase chain reaction (ddPCR) to come up with a panel of biomarkers to track ALL disease over time. The end goal of our study is to develop an assay to rapidly detect relapse and CNS disease in ALL that is more sensitive and less invasive than the current clinical assays. Earlier detection of relapse and CNS disease will provide patients with more treatment options, which may ultimately improve patient outcome. Results will ultimately be correlated with patient response to therapy, the presence of CNS disease, and overall outcomes as determined by standard clinical diagnostic procedures. Disclosures No relevant conflicts of interest to declare.

After Treatment Decrease of Bone Marrow Tregs and Outcome in Younger Patients with Newly Diagnosed Acute Myeloid Leukemia.

An emerging body of evidence demonstrates that defects in antileukemic effector cells in patients with acute myeloid leukemia (AML) can contribute to the development and/or persistence of the disease. In particular, immune suppressive regulatory T cells (Tregs) may contribute to this defective antileukemic immune response, being recruited by bone marrow leukemic cells to evade immune surveillance. We evaluated Tregs (CD4+/CD45RA-/CD25high/CD127low), performing multiparametric flow cytometry on freshly collected bone marrow aspirate (BMA), in addition to the usual molecular and cytogenetic work-up in newly diagnosed AML patients to look for any correlation between Tregs and the overall response rate (ORR). We studied 39 AML younger patients (<65 years), all treated with standard induction chemotherapy. ORR (complete remission (CR)+CR with incomplete hematologic recovery (CRi)) was documented in 21 out of 39 patients (54%); two partial responder patients were also recorded. Apart from the expected impact of the molecular-cytogenetic group (p = 0.03) and the NPM mutation (p = 0.05), diagnostic BMA Tregs did not show any correlation with ORR. However, although BMA Tregs did not differ in the study population after treatment, their counts significantly decreased in responder patients (p = 0.039), while no difference was documented in nonresponder ones. This suggested that the removal of Treg cells is able to evoke and enhance anti-AML immune response. However, the role of BMA Tregs in mediating immune system-AML interactions in the diagnostic and posttreatment phase should be confirmed in a greater number of patients.

Development and evaluation of stem cell collection procedure diagrams to support the education and recruitment of committed stem cell donors.

Diagrams which allow potential unrelated stem cell donors to visualize the stem cell collection process were hypothesized to support the recruitment and education of committed stem cell donors. A series of bone marrow and peripheral blood stem cell collection procedure diagrams were developed, featuring young adult male donors of varied ethnic backgrounds. Post-implementation, surveys were conducted to evaluate stakeholder perspective on the diagrams' utility. A quality improvement project was conducted at five stem cell drives from 2017 to 2018 at which recruiters did or did not show the diagrams to potential donors. Following the drives, registrants were invited to complete a survey exploring their experience, knowledge and attitude towards donation. The diagrams were implemented in Canada in 07/2016. Of 293 participating registrants (24·7% non-Caucasian males) recruited at five drives between 2017 and 2018, 76% (n=197) were shown the diagrams. Participants who were shown the diagrams were significantly more likely to report that the recruiters appeared very knowledgeable (89% vs. 76%, P=0·019) and to report improved self-reported knowledge of stem cell donation (P=0·010) compared to participants not shown the diagram. Data are also shown demonstrating that stakeholders in donor recruitment used and valued the diagrams and that use of the diagrams was associated with improved donor recruitment outcomes in Canada. This report is the first evaluation of stem cell collection diagrams in the literature. The diagrams are relevant to donor registries, recruitment organizations and transplant centres worldwide, and their use may support efforts to educate and recruit committed, ethnically diverse donors.

Abstract 1150: Targeting PI3K inhibits ER-mammary tumor initiation and early dissemination

Abstract Despite recent advances in cancer treatment, including targeted therapies and immunotherapies, many women still die of breast cancer. To ultimately reduce breast cancer death, it is of urgent need to develop more effective prevention and early intervention strategies. Although tamoxifen and aromatase inhibitors decrease incidences of estrogen receptor positive (ER+) breast cancer, there is no effective agent for prevention of ER negative (ER-) breast cancer. In this study, we identified that PI3K pathway is significantly activated in premalignant ER- breast lesions compared to paired normal tissue from breast cancer patients. We therefore tested whether blocking PI3K pathway by genetic knockdown or using a clinically applicable PI3K inhibitor (GDC0941) can inhibit ER- tumor initiation and progression. Indeed, both genetic knockdown of PI3K and low dose GDC0941 intervention reversed the disorganized three-dimensional growth of semi-transformed human ER- mammary epithelial cells in vitro. More importantly, low dose GDC0941 treatment significantly delayed the tumor initiation in MMTV-neu mouse model which develops ER-HER2+ mammary tumors. Notably, low dose GDC0941 treatment did not show discernable adverse effects, including no significant changes in body weight and liver functions, no immuno-toxicity. Since PI3K activation is crucial for early disseminated tumor cells, we collected bone marrow from treated mice versus vehicle control group, and examined the impact of GDC0941 treatment on early dissemination of tumor cells into the bone marrow. Excitingly, we found that administration of low dose GDC0941 significantly decreased CD45-ErBb2+/CK8/18+ early disseminated mammary tumor cells into the bone marrow, indicating that low dose PI3K inhibitor not only delayed primary tumor initiation but also prevented tumor cell early dissemination in the bone marrow. Taken together, these data support the notion that using low dose targeting drug to block PI3K pathway is safe and promising for ER- breast cancer prevention. Citation Format: Yuan Zhang, Jingyang Wang, Yi Xiao, Victoria Seewaldt, Dihua Yu. Targeting PI3K inhibits ER-mammary tumor initiation and early dissemination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1150.

Manufacturing Development of SENTI-101, a Gene Circuit Modified Allogeneic Bone Marrow Derived Mesenchymal Stromal Cell (BM-MSC) Therapy for the Treatment of Solid Tumors

Background & Aim Immunotherapies have shown success in activating endogenous immune responses against solid tumors. Many immunologically cold tumors do not respond to current immunotherapies such as checkpoint inhibitors, and present a major unmet clinical need. In prior clinical studies, allogeneic gene modified MSCs have been administered through intraperitoneal (IP) infusion to ovarian cancer patients, demonstrating safety and localization to tumor lesions.1 SENTI-101 is an allogeneic cell product consisting of MSCs engineered with a gene-circuit that expresses a potent combination of immunomodulatory cytokines: IL12 and IL21. Upon IP administration, SENTI-101 innately homes to peritoneal tumors, secretes IL12 and IL21 in a localized and sustained fashion, and induces a strong and durable anti-tumor immune response. Methods, Results & Conclusion The gene modification process for SENTI-101 includes packaging the gene circuit into a lentiviral vector, stable transduction of allogeneic BM-MSCs, expansion, and cryopreservation. The drug product is further expanded, formulated, and cryopreserved prior to clinical delivery. Key product attributes of SENTI-101 include: transduction efficiency, IL12 and IL21 secretion, cell doubling time, tumor homing, and vector copy number. A 2D culture manufacturing process was developed to maintain key product attributes over at least 14 population doublings, translating to over 10^12 final product cells per bone marrow collection. The product attributes were comparable between freshly cultured cells and those that underwent a freeze/thaw cycle and further expanded. They also showed similar localized cytokine production of IL12 and IL21 and in vivo efficacy in tumor models. Transduction efficiency was optimized by comparing different MSC donors, lentivirus titer, different chemical transduction enhancers, and other process variables. SENTI-101 was further evaluated for growth characteristics, immunomodulatory function, distribution and kinetics in mouse models, vector insertion site analysis, cell transformation assay, and karyotype. In addition, SENTI-101 was demonstrated to maintain equivalent product attributes in a 3D bioreactor process, enabling larger scale production for the allogeneic MSCs for commercial manufacturing. In conclusion, we have developed a GMP-ready process for the gene modification, expansion, and administration of SENTI-101 in preparation for upcoming human clinical studies in solid tumor indications.

Therapeutic Blood-derived stem/progenitor cells specifically activated by dendritic cells

Background & Aim Vascular diseases in which the blood supply is blocked are the main cause of death and disability globally with more people dying annually from CVD than any other disease. These diseases in people with diabetes and the elderly affects principally medium and small vessels. Stem/progenitor cell (SPCs) treatments with bone-marrow-derived cells show promising outcomes, albeit not without adverse events related to cell mobilization and bone-marrow collection. Enriched endothelial progenitor cells (EnEPCs; BGC101) generated from a patient's own standard blood draw was developed, that utilizes a one-day technology employing immune dendritic cells (DCs) to specifically direct therapeutic (SPC) activity in-vitro. Animal studies show safety and efficacy in reversing induced limb ischemia. We describe a first-in-human pilot study treating patients with scritical limb ischemia (CLI) with no surgical option. Methods, Results & Conclusion We performed a pilot open-label study of treatment with the product in patients with CLI characterized by rest pain and/or ulceration, and with no surgical option for revascularization. The product was prepared from a standard blood draw. A single treatment of the personalized cells by IM injections into the affected leg took less than 10 minutes. Acute safety follow-up was conducted during the initial 48±6 hours in the hospital and long-term follow-up at 1 week, 1, 3, 6 and 12 months. Results have exceeded the expected safety and efficacy primary endpoints. BGC101 treatment is found to be well tolerated and safe by an independent data safety board. All patients met the primary end point of amputation free survival for 6 months. A Long term herapeutic effect was seen in both objective outcomes including limb salvage, increased leg blood flow and wound healing, as well as, in subjective outcomes including, reduction of pain and usage of narcotic medications, regaining and improving walking ability and improved quality of life after a single treatment. Conclusion These observations indicate that the cell product, derived from a standard blood draw of EPC-enriched SPCs, generated by a novel culture process taking less than one day, shows promising preliminary results in the treatment of CLI. This therapy may also be therapeutic in other vascular conditions as a standalone or as adjunct therapy for other interventional procedures. The process that Further clinical studies are needed in order to test our innovative treatment in a placebo-controlled study

The Role of Autologous Mesenchymal Stem Cell Recellularization in Rescuing the Xenoreactive Immune Response

Emerging evidence suggests that current xenograft valve replacements provoke an intense cell-mediated and humoral immune response that is a major regulator of time-dependent structural valve deterioration (SVD). Autologous recellularization may improve valvular durability by masking the xenograft scaffold from immune recognition. The purpose of this study was to directly address the role of autologous recellularization of xenograft scaffolds, in the regulation of the xenoreactive immune response in an in-vitro human model. To study the human xenoreactive immune response, we collected bone marrow, human pericardium and isolated peripheral blood mononuclear cells (PBMCs) from adult patients undergoing elective cardiac surgery. Decellularized bovine pericardium underwent recellularization with human mesenchymal stem cells (MSCs). Grafts were tested as either non-decellularized (fresh) bovine pericardium, decellularized bovine pericardium and recellularized bovine pericardium. We show that when decellularized bovine pericardium was recellularized with human MSCs and exposed to autologous human PBMCs in-vitro, there was a significant reduction in TNF-α and IFN-γ expression, along with a significant decrease in T-cell proliferation when compared to both wild-type and decellularized bovine pericardium (p<0.01). Importantly, recellularized bovine pericardium had an equivalent expression of TNF-α, IFN-γ and T-cell proliferation when compared to human pericardium exposed to autologous human PBMCs. Our data suggest that autologous MSC recellularization of xenogenic tissue abrogates the xenoreactive immune response with subsequent reduction in pro-inflammatory cytokine production and T-cell proliferation in an in-vitro human model. As such, autologous MSC recellularization of an acellular xenogenic scaffolds may be an effective approach to decrease the progression of bioprosthetic SVD.

Clonal selection in therapy-related myelodysplastic syndromes and acute myeloid leukemia under azacitidine treatment.

Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/AML) are defined as complications of previous cytotoxic therapy. Azacitidine (AZA), a hypomethylating agent, has showed activity in t-MDS/AML. We evaluated the clonal dynamics of AZA-treated t-MDS/AML. We collected bone marrow samples, at diagnosis and during treatment, from AZA-treated t-MDS/AML patients. NGS on 19 myeloid genes was performed, and candidate mutations with a variant allele frequency >5% were selected. Seven t-AML and 12 t-MDS were included with median age of 71 (56-82) years old, median number of AZA cycles of 6 (1-15), and median overall survival (OS) of 14 (3-29) months. We observed correlation between AZA response and clonal selection. Decrease of TP53-mutated clone was correlated with response to AZA, confirming AZA efficacy in this subgroup. In some patients, emergence of mutations was correlated with progression or relapse without impact on OS. Clones with mutations in genes for DNA methylation regulation frequently occurred with other mutations and remained stable during AZA treatment, independent of AZA response. We confirmed that the molecular complexity of t-MNs and that the follow-up of clonal selection during AZA treatment could be useful to define treatment combination.

Efficacy and Safety of Sedo-Anesthesia in Autologous Stem Cells Transplantation in Children with Cerebral Palsy or Autism: Propofol Following Sevoflourane Inhalation

Introduction Intrathecal autologous bone marrow (ABM) total nucleated cells transplantation (TNCT) has been reported to be a promising treatment for cerebral palsy (CP) and autistic disorders (AD). Children with CP and AD, are expected to be patients with difficult approach when doing venipuncture or taking them to the operating room. Considering pain and the nature of the procedures, bone marrow collection (BMC) and intrathecal procedures (ITP) must be performed under sedation. For short procedures, sevoflurane can be administrated using a face mask and, propofol, a typical intravenous agent is used for the induction of general anesthesia and for sedation. Objective Evaluate the safety and efficacy of sedation performed by anesthesiologists, prior to BMC and ITP. Methods Retrospective study of 97 pediatric patients who underwent BMC and further lTP for TNCT under sedation and local anesthesia in a hospital of a developing country from January 2018 to February 2019. Prior to the procedures, patients were fasted for 8 hours. Anesthesia was administered to each patient at two times, once for BMC and a second for ITP of TNC. For the first induction, patients received inhaled sevoflurane by anesthetic planes, with an oxygen flow of 2 L/min. When Ramsay scale was scored 6, venous access was installed and used for the second induction as well. For the ITP, each child was intravenously injected with 2mg/kg of propofol; supplemental doses of 1 mg/kg were repeatedly given when sedation level was not reached. Severe adverse events were defined as cardiovascular collapse: heart rate (HR) Results Where included 97 patients, resulting in 194 anesthesic procedures, 97 for BMA and 97 for LP. Demographic data are shown in Figure I. Median propofol administration was 40 (24.5-56) ml (Figure I). Post hoc analysis were performed, reporting differences between first and second, as well as second and third systolic blood pressure (BPsys), SpO2, HR and RR evaluations (p Conclusions Sevoflurane-propofol combinations for children with CP and AD resulted in stable hemodynamic response and few adverse effects and may be suitable for the induction of short-term anesthesia during BMC and IT TNCT.

Stem cells out of the bag: characterization of ex vivo expanded mesenchymal stromal cells for possible clinical use.

Aim:Mesenchymal stromal cells (MSC) are a promising tool for cellular therapy and regenerative medicine. One major difficulty in establishing a MSC expansion protocol is the large volume of bone marrow (BM) required. We studied whether cells trapped within a collection bag and filter system could be considered as a source of MSC.Results:From the 20 BM collection bag and filter systems, we recovered an average of 1.68 × 108 mononuclear cells, which is the equivalent to 60 ml of filtered BM. Mononuclear cells were expanded ex vivo to 17 × 106 MSC, with purity shown by a CD44+, CD105+, CD90+ and CD73+ immunophenotype, a reduction of 20% proliferating cells in a mixed lymphocyte reaction and also the ability of adipocyte differentiation.Conclusion:Long-term MSC cultures were established from the usually discarded BM collection bag and filter, maintaining an appropriate phenotype and function, being suitable for both investigation and clinical settings.

Possibility of using the infrapatellar (Hoffa's) fat pad as a source of autologous stem cells

Stem cells are the basis for the creation of tissue-engineered structures in regenerative medicine. The most well-studied sources of stem cells are the embryo and bone marrow. The use of embryonic cells is associated with ethical problems, and the collection of bone marrow is accompanied by invasive procedures. Using adipose tissue as a source of stem cells avoids these problems. But the collection of adipose tissue requires additional interventions, which does not exclude the occurrence of cosmetic defects. Aim of the study was to investigate the possibility of using mesenchymal stem cells (MSCs) isolated from the infrapatellar (Hoffa’s) fat pad. Material and methods . As a source of MSCs, tissue samples of Hoffa’s fat pad removed during the operation were used (8 cases), as a control - MSCs isolated from human adipose tissue (6 cases). MSCs were isolated using an enzymatic method. At the 3rd passage, phenotyping with specific antibodies against CD34, CD45, CD73, CD90, CD105 was performed by flow cytometry. Differentiation in the chondro- and osteogenic direction was carried out at the 3rd passage with the appropriate differentiation media. Chondrogenic differentiation was confirmed by staining with alcian blue, osteogenic - staining according to von Kossa. Results and discussion . Statistically significant decrease in CD105 expression, increase in CD73, CD34 expression and lack of adequate differentiation under standard conditions of differentiation media by MSCs isolated from the Hoffa’s fat pad compared to control was found. The data obtained indicate a discrepancy between the cells isolated from the Hoffa’s fat pad and the requirements for MSCs. Conclusion . The infrapatellar (Hoffa’s) fat pad_cannot be used as a source of standardized MSCs.

Experimental assessment of permethrin-fipronil combination in preventing Leishmania infantum transmission to dogs under natural exposures

Canine vector-borne disease transmission can be reduced with regular use of repellent insecticides. The objective of this year-long experimental study was to assess the efficacy of a topical formulation of fipronil/permethrin (Frontline Tri-Act®) in preventing transmission of Leishmania infantum by sandflies. This clinical field trial was conducted in Xanthi (Northern Greece), an area highly endemic for canine leishmaniosis, from April 2018 to March 2019. Forty purpose-bred Beagle dogs, testing negative for L. infantum prior to study initiation, were enrolled in the study, which consisted of three phases: Phase 1 (field exposure phase) took place from Day 0–196. The dogs were randomly allocated to two groups, group 1 (sham-treated topically with sterile water) and group 2 (treated topically with Frontline Tri-Act®). Dogs in both groups were housed in two subunits of an open-air kennel for a period of 7 months, spanning the Leishmania transmission season. All dogs were treated or sham-treated on Days 0, 28, 56, 84, 112, 140 and 168. Clinical examinations, PCR analysis of conjunctival swabs, and serological tests were performed on a monthly basis. Sandflies were collected every 2 weeks, during a 12-h period overnight using light traps. Each collection was placed in a container and kept refrigerated until speciation and PCR analysis could be performed. In the second phase of the study, from Day 197 to 252, the dogs were moved into a protected environment (insect-proof protected environment phase). CDC light traps were activated every 2 weeks inside and outside the kennels to ensure the vector-free status of the facility. Monthly clinical examinations, including PCR analysis of conjunctival swabs, and serological tests continued. At the end of the phase 2, bone marrow samples were collected on all dogs. Phase 3 (the final post-winter check) took place from Day 253 to 350. Dogs were released and adopted by individual private owners on Day 253. Follow up analyses included blood collection for SNAP tests and conjunctival swaps for PCR analysis on Days 304 and 350. Additionally, bone marrow collections were also performed on Day 350.Presence of sandflies was observed only in the phase 1 exposure period, and 1714 sandflies were collected (1427 females and 287 males). Two species were identified, Phlebotomus perniciosus var. tobbi and Phlebotomus neglectus. Out of the 62 pooled samples of sandflies assessed by PCR, three were considered positive (4.8 %). By the end of the study, 35 % of the Group 1 dogs (7/20) became positive based on PCR (conjunctival swab and bone marrow) and 30 % (6/20) based on SNAP/IFAT and ELISA tests, while all the dogs in the Frontline Tri-Act® treated group 2 remained negative for all tests (G1 vs G2, p=0.008). All tests identified the same positive animals, and PCR allowed the detection of one additional infected dog. This clinical field trial demonstrated that monthly administration of Frontline Tri-Act® to dogs exposed to Leishsmania infection in a high endemic area provided 100 % preventive efficacy against transmission of L. infantum.

Association between Characterizations of Bone Marrow Mesenchymal Stem Cells, Ejection Faction and Hospitalization Period in Patients with Severe Left Ventricular Dysfunction after Off-Pump Bypass Surgery

Background and Aim: Severe left ventricular dysfunction patients with ejection fraction ≤ 30% are at a higher risk for complications and mortality than patients with ejection fraction >30%. The death of cardio-myocytes at injured regions leads to myocardium dysfunction. Bone marrow-derived mesenchymal stem cells are undifferentiated cells that have been used for the regeneration of damaged cardio-myocytes. Due to the inherent capability of mesenchymal stem cells to improve cardiac functions, in this research, our objective was to explore the possible association of the mesenchymal stem cells proliferation rate with Coronary Artery Bypass Grafting outcomes in patients with severe left ventricular dysfunction after off-pump Coronary Artery Bypass Grafting. Methods: For investigating the possible association of mesenchymal stem cells proliferation with Coronary Artery Bypass Grafting outcomes (ejection fraction, hypertension risk, and the time of hospital stay), we collected bone marrow samples from 30 patients (18 men and 12 women) who underwent off-pump Coronary Artery Bypass Grafting at Afshar Hospital and Seyed Al-Shohada Hospital (Yazd, Iran). Mesenchymal stem cells were isolated and cultured; then, cells were counted after 4, 7, and 14 days using trypan-blue color, and doubling times were calculated. Results: There was an association between doubling time and ejection fraction after surgery. Ejection fraction in postoperative patients increased, but this association was not significant. Also, our study showed that the risk of hypertension is equal in male and female patients. There were no significant differences in mesenchymal stem cells doubling time with hypertension and the time of hospital stay in the ICU. Conclusion: Based on this study, we concluded that there was no significant relationship between the rate of mesenchymal stem cell proliferation and Coronary Artery Bypass Grafting outcomes in patients with severe left ventricular dysfunction. *Corresponding Author: Zakieh Sadat Sheikhalishahi; Email: z.sheikhalishahi@gmail.com Please cite this article as: Esmailidehaj M, Mirhosseini SJ, Sheikhalishahi ZS. Association between Characterizations of Bone Marrow Mesenchymal Stem Cells, Ejection Faction and Hospitalization Period in Patients with Severe Left Ventricular Dysfunction after Off-Pump Bypass Surgery. Arch Med Lab Sci. 2019;5(4):30-36. https://doi.org/10.22037/amls.v5i4.31296

Characterization of progenitor/stem cell population from human dental socket and their multidifferentiation potential.

Dental stem cells have many applications in medicine, dentistry and stem cell biology in general due to their easy accessibility and low morbidity. A common surgical manoeuvre after a tooth extraction is the dental socket curettage which is necessary to clean the alveolus and favour alveolar bone healing. This procedure can cause very low morbidity compared to bone marrow collection procedures and the collected material is normally discarded. In order to investigate if the tissue obtained by dental socket curettage after a tooth extraction was a feasible alternative source to isolate human stem cells, we isolated and characterized two different stem cell populations based on STRO-1 and CD146 expression. We were able to collect and grow cells from dental socket of vital and non-vital teeth. Both populations were proliferative, clonogenic and expressed STRO-1, CD146, CD90, NG2, PDGFR-β, which are markers found in stem cells, presented in vitro multiline-differentiation into osteogenic, chondrogenic, and adipogenic tissue, and in vivo transplanted cells formed mineralized tissue. Interestingly, STRO-1+ clonogenic cells presented better multidifferentiation than CD146+ cells. Our results showed that mesenchymal stem cells can be isolated from the tiny tissue collected by dental socket curettage after vital and non-vital tooth extraction and suggest that STRO-1 is an important marker to be used to sort cells with multidifferentiation capacity.

Collection and Processing of Bone Marrow at 3% Oxygen Significantly Alters the Manifestation of Aged Mouse Hematopoietic Stem Cell Phenotype

Aging is an inevitable process associated with eventual deterioration of normal physiological functions. Aged hematopoiesis is associated with increased numbers of hematopoietic stem cells (HSC), but with decreased HSC functional activity (e.g. decreased engrafting capability in lethally irradiated mice and a shift in the myeloid:lymphoid bias of the engrafting HSC of the old mice, such that there are more myeloid but fewer lymphoid cells generated from HSC of the old mice). Production of HSC and progenitor (HPC) cells ex vivo is more efficient when cells are cultured in a hypoxic environment of ~ 5% oxygen tension than when cells are grown at ambient air (~21% oxygen). The bone marrow (BM) microenvironment niche that nurtures the survival and production of HSC and HPC and hematopoiesis during adult life is a hypoxic environment (~1-5% oxygen tension) compared to that of ambient air. However, almost all results of studies of young and aged mouse hematopoiesis have been based on numbers and activity of HSC and HPC that have been collected and processed in ambient air. Our recent work evaluating hematopoiesis in BM cells of young adult mice and with human cord blood cells found, through a phenomenon we designated Extra Physiological Oxygen Shock/Stress (EPHOSS), that there is a large loss of HSC with an increase of HPC within minutes of the collection of these cells in ambient air (Mantel et al., Cell, 2015). This led us to reason that perhaps what we know about aging hematopoiesis might not be entirely accurate and that a re-evaluation of aged HSC, HPC, and hematopoiesis was in order. We hypothesized that hematopoiesis in aged (~20-27 months of age) mice may not be as dysregulated as reported but that collection and processing of BM from the aged mice is more sensitive than similar cells from young (~6-16 weeks) mice to EPHOSS-induced events generated by the collection of the cells in ambient air. We evaluated BM from three different mouse strains (CB6, BALB/c, and C57Bl/6) at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or hypoxia (3% oxygen). BM from old mice collected/processed under hypoxic conditions exhibited phenotypically increased long-term HSC and common lymphoid progenitor (CLP) numbers and decreased common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) numbers when compared to old BM collected/processed under ambient air conditions. BM collected from old C57Bl/6 mice under hypoxia had increased engrafting capability more closely matching that of young BM. This was associated with a 3.14-fold increase in the number of competitive repopulating units (representative of functional HSC) in old BM collected under hypoxic conditions compared to old BM collected in ambient air as determined through limiting dilution analysis. The myeloid:lymphoid ratio of old BM collected under hypoxia matched that of young BM collected under air. This was associated with decreased cycling of CFU-GM, BFU-E, and CFU-GEMM in old BM collected/processed in hypoxia. Enhanced numbers/function of old BM HSCs collected in hypoxia is associated with changes in expression of CXCR4 (and HSC homing capability), CCR5, stress protein levels (e.g. HSP40 etc) and ROS (both total and mitochondrial). All of these noted changes demonstrated that the old BM collected/processed under hypoxic conditions more closely resemble functionally young BM. Thus, age-related differences between the HSC/HPC populations are not as drastic as previously reported and reflect the increased sensitivity of hematopoiesis from aged mice to an artificial ambient air collection procedure. Disclosures No relevant conflicts of interest to declare.

Dose-Adjusted Granulocyte Colony-Stimulating Factor Plus Dexamethasone Achieves More CD34+ Cell Mobilization and Earlier Engraftment in Haploidentical Hematopoietic Stem Cell Transplantation

Introduction: Previous studies have showed that higher doses of CD34+ cell were associated with more rapid neutrophil and platelet engraftment, lower probabilities of graft rejection, as well as reduced transplant-related mortality. The aim of this study was to investigate the effects of G-CSF (Filgrastim) plus dexamethasone in CD34+ cell mobilization and engraftment in T-cell replete haploidentical hematopoietic stem cell transplantation(HHSCT) which was based on G-CSF-primed bone marrow and peripheral blood graft. Methods: A total of 79 healthy donors, who underwent bone marrow (BM) harvest and peripheral blood Stem Cells (PBSCs) collection between January 2015 and June 2019, were investigated. In G-CSF group, G-CSF was administered subcutaneously at a dose of 5μg/kg once a day from 1st to 5th day, while BM and PBSC were harvested on the 4th day and 5th day, respectively. In Dose-Adjusted G-CSF+Dex group, G-CSF was administered subcutaneously at a dose of 5μg/kg once a day on the 1st and 2nd day, then twice a day from the 3rd to 5th day; 5mg dexamethasone was injected intravenously before BM collection on the 4th day and before PBSC apheresis on the 5th day, respectively. All 79 recipients with hematological malignancies underwent HHSCT based on modification of BU/CY (busulfan/cyclophosphamide) and Anti-human T Lymphocyte Rabbit Immunoglobulin (ATG-F). All recipients received cyclosporine A, mycophenolate mofetil, and short-term cyclophosphamide as GVHD prophylaxis. Results: There were no significantly statistical differences between these two groups on characteristics of both recipients and donors. In Dose-Adjusted-G-CSF+Dex group, more mono nuclear cells (MNCs) were collected from BM and PB in comparison to the cells collected in the G-CSF group (p&lt;0.001). There was a significant difference between the two groups on CD34+ cell counts from PB (p=0.002), which led to a significant difference on CD34+ cells in mixture allografts (p=0.04). In DA-G-CSF + Dex group, more CD34+ cells achieved earlier neutrophil (p=0.001) and platelet (p&lt;0.001) engraftment compared with G-CSF group. Conclusion: Compared to G-CSF alone, dose-adjusted G-CSF plus dexamethasone on healthy donors can lead to more collection of MNCs and CD34+ cells in mixture allografts, which achieves earlier neutrophil and platelet engraftment. Disclosures Zheng: Pfizer: Research Funding.

Sequencing of Circulating Cell-Free DNA in Patients with AML Detects Clinically Significant Mutations Not Detected in Bone Marrow: The Role for Complementary Peripheral Blood and Bone Marrow Genomic Analysis

Sequencing of Circulating Cell-Free DNA in Patients with AML Detects Clinically Significant Mutations Not Detected in Bone Marrow: The Role for Complementary Peripheral Blood and Bone Marrow Genomic Analysis