Fibroblast Colony-forming Units Research Articles

Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods.

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

Xeno-Free Condition Enhances Therapeutic Functions of Human Wharton's Jelly-Derived Mesenchymal Stem Cells against Experimental Colitis by Upregulated Indoleamine 2,3-Dioxygenase Activity.

The therapeutic applications of mesenchymal stem cells (MSCs) have been actively explored due to their broad anti-inflammatory and immunomodulatory properties. However, the use of xenogeneic components, including fetal bovine serum (FBS), in the expansion media might pose a risk of xenoimmunization and zoonotic transmission to post-transplanted patients. Here, we extensively compared the physiological functions of human Wharton’s jelly-derived MSCs (WJ-MSCs) in a xeno-free medium (XF-MSCs) and a medium containing 10% FBS (10%-MSCs). Both groups showed similar proliferation potential; however, the 10%-MSCs showed prolonged expression of CD146, with higher colony-forming unit-fibroblast (CFU-F) ability than the XF-MSCs. The XF-MSCs showed enhanced adipogenic differentiation potential and sufficient hematopoietic stem cell (HSC) niche activity, with elevated niche-related markers including CXCL12. Furthermore, we demonstrated that the XF-MSCs had a significantly higher suppressive effect on human peripheral blood-derived T cell proliferation, Th1 and Th17 differentiation, as well as naïve macrophage polarization toward an M1 phenotype. Among the anti-inflammatory molecules, the production of indoleamine 2,3-dioxygenase (IDO) and nitric oxide synthase 2 (NOS2) was profoundly increased, whereas cyclooxygenase-2 (COX-2) was decreased in the XF-MSCs. Finally, the XF-MSCs had an enhanced therapeutic effect against mouse experimental colitis. These findings indicate that xeno-free culture conditions improved the immunomodulatory properties of WJ-MSCs and ex vivo-expanded XF-MSCs might be an effective strategy for preventing the progression of colitis.

Human umbilical cord stem cells – the discovery, history and possible application

Abstract Stem cells are now one of the most extensively studied fields in experimental medicine. Although embryonic stem cells (ESCs) have the capability to differentiate into lineages derived from all three primary germ layers, their use remains ethically controversial. Mesenchymal stromal cells’ (MSCs) utilization is not burdened with such concerns. MSCs were primarily characterized as fibroblastic cells from bone marrow stroma, with the ability to adhere to plastic and form clonogenic colonies (CFU-Fs – colony-forming unit-fibroblasts). Subsequent studies led to their isolation from various other adult or perinatal tissues, including the umbilical cord. Although in most cases MSCs are obtained from umbilical cords’ stroma (Wharton’s jelly), it has been demonstrated that both cord’s lining and endothelial layer of umbilical vein possess the cells that fulfill the minimal criteria for MSCs published in 2006 by the International Society for Cellular Therapy. Since the initial discovery, a number of pre-clinical and clinical studies were conducted with the use of MSCs derived from umbilical cords, targeting diseases such as cancer or autoimmune diseases. Moreover, their secretome may be even more beneficial, providing an opportunity to create cell-free therapies. Running title: The history of human umbilical cord stem cells

Mesenchymal stem cells – a historical overview

Abstract Mesenchymal stem cells are currently one of the most extensively studied topics in experimental medicine, given their unique properties in terms of immunomodulation, multiple factors secretion and homing to injured tissue sites. Such characteristics were proven to be invaluable in various disease management treatments, for example in cancer, tissue regeneration or immunologic/inflammatory-related disorders. MSCs were first isolated from bone marrow in 1960-1970’s and were characterized as cells with fibroblastoid shape and osteogenic potential, which form clonogenic colonies (CFU-F – colony-forming unit-fibroblast). Nowadays the term ‘mesenchymal stem cells’ is used in regards to all of the cells meeting minimal criteria published in 2006 by the International Society for Cellular Therapy, however the name ‘mesenchymal stromal cells’ has been suggested to be more appropriate. Regardless of the name controversy, these cells exhibit multilineage differentiation potential, self-renewal ability, adhere to plastic and express specific surface antigens. In 2011 the first commercial product based on MSCs was developed and many more are expected to emerge. This review focuses on a historical perspective concerning studies on MSCs, controversies regarding their name and their characteristics and clinical utilization. Running title: The history of mesenchymal stem cells

A Comparative Quantification in Cellularity of Bone Marrow Aspirated with two New Harvesting Devices, and The Non-equivalent Difference Between A Centrifugated Bone Marrow Concentrate And A Bone Marrow Aspirate As Biological Injectates, Using A Bi-Lateral Patient Model

The first aim of this study was to examine the cellularity and quality of autologous bone marrow aspirates harvested with two novel FDA-cleared devices, namely the Aspire™ bone marrow aspiration system (AS-BMAS) and the Marrow Cellution bone marrow aspiration device (MC-BMAD). Compared to traditional bone marrow harvesting needle systems, both these devices have a closed distal tip, avoiding preferential marrow collection (peripheral blood aspiration) from deeper cavity regions, whereas the side holes facilitate more horizontal marrow extraction. In all patients, a similar harvesting technique was used. The second aim was to demonstrate the effectiveness of mechanical centrifugation of a large volume of extracted bone marrow to produce a bone marrow concentrate (BMC). Finally, we directly compared bone marrow constituents aspirated with MC-BMAD with a BMC, generated by centrifugation of bone marrow harvested using the AS-BMAS. A bi-lateral patient model was used for all comparisons. All cellular analyses included the measurement of Colony-Forming Units-fibroblasts (CFU/f) levels, CD34+cells/ml, Total Nucleated Cells (TNCs)/ml, platelets/ml, and Red Blood Cells (RBCs)/ml in a single, FDA-approved laboratory, compliant with Good Manufacturing Practice regulations. A total of 12 patients consented to the study. In the direct BMA comparison, the AS-BMAS bone marrow yielded significantly higher CFU/f counts and TNC concentrations than the MC-BMAD (1,060/ml, 33.5 × 106/ml, and 610/ml and 28.6 × 106/ml, respectively), with comparable platelet and RBC concentrations. Data following BMA concentration to produce a BMC revealed highly significant cell yields, fewer RBCs, and lower hematocrit (HCT). A direct cellular comparison between the aspirate of the MCBMAD and centrifugated BMC following AS-BMAS marrow extraction showed highly significant differences in cellularity. The AS-BMAS produced cell concentrations of CFU/f, CD34+ cells, TNCs, platelets, and RBCs that were comparable, or greater than, the predicate device. We believe that concentrating bone marrow is a consistent and safe method to produce a BMC that has the potential to be clinically effective. Furthermore, data indicate a non-equivalent difference in BMC cellularity, when compared to a non-filtered and non-centrifugated BMA for clinical use.

Quantification and Qualification of Stem Cells From Blood After Mobilization With Filgrastim, and Concentration Using a Platelet-Rich Plasma System

To determine the cellular composition of a product created with peripheral blood harvested after systemic mobilization with filgrastim and processed with one point-of-care blood concentrating system, i.e., a platelet-rich plasma (PRP) system. The second purpose was to compare mobilized platelet-rich plasma (M-PRP) with a concentrated bone marrow aspirate (cBMA) and a PRP created from the same subjects with the same PRP system. Ten healthy volunteer subjects were recruited for collection and analysis of 3 tissue sources: non-treated peripheral blood, bone marrow aspirate, and filgrastim-mobilized peripheral blood, involving 4 doses of weight-based filgrastim. One point-of-care blood and bone marrow concentrating system was used to create 3 products: PRP, cBMA, and M-PRP. Automated hematologic analysis was performed on all products to quantify total red blood cells, white blood cells (WBCs), monocyte, platelet, and hematopoietic progenitor cell (HPC) concentrations. Flow cytometry was used to determine hematopoietic and mesenchymal progenitor cell populations. Lastly, concentrates were cultured and fibroblast colony-forming units (CFU-F) and morphology of adherent cells were evaluated. M-PRP contained a greater concentration of WBC (mean difference= 53.2 k/μL; P < .0001), monocytes (mean difference= 8.3 k/μL; P= .002), and a trend toward a greater concentration of HPC (mean difference= 200.5 /μL; P= .060) when compared with PRP. M-PRP contained a greater concentration of monocytes (mean difference= 5.5 k/μL; P= .017) and a trend toward a greater concentration of platelets (mean difference= 348 k/μL; P= .051) and HPC (mean difference= 193.4 /μL; P= .068) when compared with cBMA. M-PRP had a similar concentration of platelets to PRP (mean difference= 110 k/μL; P= .051) and PRP had a greater concentration than cBMA (mean difference= 458 k/μL; P= .003). cBMA remained the only product capable of producing CFU-Fs (446 ± 247 /mL) as neither the M-PRP nor PRP produced CFU-Fs. M-PRP produced colonies consistent with WBC. M-PRP, produced with filgrastim mobilized blood and a proprietary PRP system, contained more total WBCs, monocytes, platelets, and HPCs than cBMA and more WBCs, monocytes, and HPCs than PRP. Filgrastim mobilized PRP may be an alternative to cBMA for use as a point-of-care product for orthopaedic treatments.

Overexpression of GATA4 enhances the antiapoptotic effect of exosomes secreted from cardiac colony-forming unit fibroblasts via miRNA221-mediated targeting of the PTEN/PI3K/AKT signaling pathway

BackgroundGATA4 is an early cardiac-specific transcription factor, and endogenous GATA4-positive cells play a critical role in cardioprotection after myocardial injury. As functional paracrine units of therapeutic cells, exosomes can partially reproduce the reparative properties of their parental cells. Here, we investigated the cardioprotective capabilities of exosomes derived from cardiac colony-forming unit fibroblasts (cCFU-Fs) overexpressing GATA4 (cCFU-FsGATA4) and the underlying mechanism through which these exosomes use microRNA (miRNA) delivery to regulate target proteins in myocardial infarction (MI).MethodsExosomes were harvested from cCFU-Fs by ultracentrifugation. miRNA arrays were performed to determine differential miRNA expression between exosomes derived from cCFU-FsGATA4 (GATA4-Exo) and control cCFU-Fs (NC-Exo). A dual-luciferase reporter assay confirmed that miR221 directly targets the 3′ untranslated region (UTR) of the phosphatase and tensin homolog on chromosome ten (PTEN) gene. Cardiac function and myocardial infarct size were evaluated by echocardiography and Masson trichrome staining, respectively.ResultsCompared with NC-Exo, GATA4-Exo increased the survival and reduced the apoptosis of H9c2 cells. Direct intramyocardial transplantation of GATA4-Exo at the border of the ischemic region following ligation of the left anterior descending (LAD) coronary artery significantly restored cardiac contractile function and reduced infarct size. Microarray analysis revealed significantly increased miR221 expression in GATA4-Exo. qPCR confirmed higher miR221 levels in H9c2 cells treated with GATA4-Exo than in those treated with NC-Exo. miR221 mimic-transfected H9c2 cells demonstrated a significantly higher survival rate following exposure to hypoxic conditions than those transfected with miR221 inhibitor. A dual-luciferase reporter gene assay confirmed the PTEN gene as a target of miR221. Western blot analysis showed that H9c2 cells treated with GATA4-Exo exhibited lower PTEN protein expression and higher p-Akt expression.ConclusionGATA4 overexpression enhances the protective effect of cCFU-F-derived exosomes on myocardial ischemic injury. In terms of the mechanism, it is at least partly due to the miR221 transferred by GATA4-Exo, which inhibits PTEN expression, activates the phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway, and subsequently alleviates apoptosis of myocardial cells (CMs).

Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay

PurposeThe prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification. MethodsFreshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density. ResultsA nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used. ConclusionsFreshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.

Human Mesenchymal Stromal Cell (MSC) Characteristics Vary Among Laboratories When Manufactured From the Same Source Material: A Report by the Cellular Therapy Team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

BackgroundCulture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods and different source materials. The purpose of this study was to assess the impact on MSC characteristics when different laboratories propagated MSCs from cultures initiated with BM aliquots derived from the same donor source material.Methods and MethodsFive aliquots from each of three different BM donors were distributed to five independent laboratories. Three laboratories plated whole BM and two laboratories a mononuclear BM cell fraction. Four laboratories cultured in media supplemented with fetal bovine serum (FBS) and one laboratory used human platelet lysate (hPL). Initial cell seeding densities (i.e., P0) ranged from 19.7 × 103/cm2–282 × 103/cm2 and for second seeding (i.e., P1) 0.05 × 103–5.1 × 103 cells/cm2. Post-thawed MSCs from each laboratory were analyzed for cell viability, immunophenotype, tri-lineage differentiation, fibroblast colony-forming units (CFU-F), gene expression, and immunosuppressive activity.ResultsTransit times from BM collection to receipt by laboratories located in the United States ranged from 16.0–30.0 h and from 41.5–71.5 h for a laboratory in Asia. Post-thaw culture derived MSCs rom BM #1, #2, and #3 exhibited viabilities that ranged from 74–92%, 61–96%, and 23–90%, respectively. CFU activity from BM #1, #2, and #3 per 200 MSCs plated averaged 45.1 ± 21.4, 49.3 ± 26.8 and 14.9 ± 13.3, respectively. No substantial differences were observed in immunophenotype, and immunosuppressive activities. Global gene expression profiles of MSCs revealed transcriptome differences due to different inter-laboratory methods and to donor source material with the center effects showing greater molecular differences than source material.ConclusionFunctional and molecular differences exist among MSCs produced by different centers even when the same BM starting material is used to initiate cultures. These results indicated that manufacturing of MSCs by five independent centers contributed more to MSC variability than did the source material of the BM used in this study. Thus, emphasizing the importance of establishing worldwide standards to propagate MSCs for clinical use.

Isolation of Amniotic Fluid Mesenchymal Stem Cells Obtained from Cesarean Sections

BACKGROUND: The term amniotic fluid (AF) can be an ideal alternative as a source of mesenchymal stem cells (MSCs), originating from the neonate. Preclinical studies of the second- and third-trimester amnion fluid cells confirmed the number of potential donors from this wasted material.&#x0D; AIM: This study aims to look at the forming time of stem cells derived from AF-MSCs.&#x0D; MATERIALS AND METHODS: AF samples from healthy human donors were collected during full-term C-sections and kept at 4°C until processed. The number of colony-forming unit-fibroblast was assessed microscopically by calculating spindle-shaped colonies that clearly resembled of fibroblasts and did not include colonies with rounded epithelioid morphology. The immunophenotyping of their independent AF preparations was done using the human MSC phenotyping kit which was done according to the manufacturer’s instruction by flow cytometry.&#x0D; RESULTS: The result showed that it succeeded in getting 8 million cells which will be used for research on pelvic organ prolapse therapy using AF-MSCs. The stem cell isolation totally takes 6 weeks. We got 2 million stem cells in one flask.&#x0D; CONCLUSION: This study concludes that the time needed for differentiation of AF-MSCs is 6 weeks and AF-MSCs express mesenchymal markers such as CD90, CD73 (SH3, SH), and CD105 (SH2) and these cells also express HLA antigens – ABC, CD 34, and CD 45 which are hematopoietic markers and endothelial CD31 markers.

Dimethyloxalyl Glycine Regulates the HIF-1 Signaling Pathway in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multidirectional differentiation capabilities. Dimethyloxalyl glycine (DMOG) mobilizes MSCs, and the hypoxia inducible factor-1 (HIF-1) signaling pathway plays an important role in MSC mobilization. We aimed to investigate the effect of DMOG on the HIF-1 pathway in MSCs. Rats were treated with DMOG, and the numbers of peripheral blood MSCs (PB-MSCs) and bone marrow MSCs (BM-MSCs) were detected by the Colony-forming unit fibroblastic (CFU-F) method. The growth curve, cell cycle and migration ability of PB-MSCs and BM-MSCs were detected by CCK-8, Flow cytometry and Transwell assays. Western blotting and real-time qPCR were used to detect the expression of the HIF-1 pathway. The number of bone marrow microvessels was detected by immunohistochemistry. DMOG significantly increased the numbers of PB-MSCs and BM-MSCs (P < 0.05). Further, the MSCs in peripheral blood and bone marrow still had the ability to proliferate and migrate after mobilization by DMOG. The expression levels of HIF-1α, stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) in MSCs were significantly regulated by DMOG (P < 0.05). The number of bone marrow microvessels decreased after the VEGF/VEGFR signaling pathway was blocked by SU5416 (P < 0.05). Therefore, these findings demonstrated that DMOG regulates the HIF-1α signaling pathway and promotes biological effects in MSCs.

Comprehensive analysis of skeletal muscle- and bone-derived mesenchymal stem/stromal cells in patients with osteoarthritis and femoral neck fracture

BackgroundMesenchymal stem/stromal cells (MSCs) can replenish the aged cells of the musculoskeletal system in adult life. Stem cell exhaustion and decrease in their regenerative potential have been suggested to be hallmarks of aging. Here, we investigated whether muscle- and bone-derived MSCs of patients with osteoarthritis and osteoporosis are affected by this exhaustion, compared to healthy donors.MethodsPatients with primary osteoarthritis, femoral neck fractures due to osteoporosis, and healthy donors (controls) were included. MSCs were isolated from the skeletal muscle and subchondral bone from each patient and compared using ex vivo and in vitro analyses, including immunophenotyping, colony-forming unit fibroblast assays, growth kinetics, cell senescence, multilineage potential, and MSC marker gene expression profiling.ResultsFreshly isolated cells from muscle from patients with osteoarthritis showed a lower proportion of CD45/CD19/CD14/CD34-negative cells compared to patients with osteoporosis and healthy donors. Freshly isolated muscle cells from patients with osteoarthritis and osteoporosis also showed higher clonogenicity compared to healthy donors. MSCs from both tissues of osteoarthritis patients showed significantly reduced osteogenesis and MSCs from the bone also reduced adipogenesis. Chondrogenic pellet diameter was reduced in bone-derived MSCs from both patient groups compared to healthy donors. A significant positive correlation was observed between adipogenesis and CD271 expression in muscle-derived MSCs. CD73 was significantly lower in bone-derived MSCs from osteoarthritis patients, compared to osteoporosis patients. Gene expression profiling showed significantly lower expression of MSC marker gene leptin receptor, LEPR, previously identified as the major source of the bone and adipocytes in the adult bone marrow, in bone-derived MSCs from patients with osteoarthritis in comparison with osteoporotic patients and healthy donors.ConclusionsOur results show deficient ex vivo and in vitro properties of both skeletal muscle- and bone-derived MSCs in osteoarthritis and osteoporosis patients, compared to healthy donors. In bone-derived MSCs from patients with osteoarthritis, we also identified a lower expression of the leptin receptor, a marker of MSCs that present a major source of MSCs in the adult bone marrow. This suggests that exhaustion of skeletal muscle- and bone-derived MSCs is a hallmark of osteoarthritis and osteoporosis, which defines the need for further clinical trials of stem cell transplantation in these patients.

Transient Existence of Circulating Mesenchymal Stem Cells in the Deep Veins in Humans Following Long Bone Intramedullary Reaming.

The biology of mesenchymal stem cells (MSCs) in humans is incompletely understood and a possible role of systemically circulating cells in health and autoimmune disease remains controversial. Physiological movement of bone marrow MSCs to sites of injury would support the rationale for intravenous administration for relocation to damaged organs. We hypothesized that biophysical skeletal trauma rather than molecular cues may explain reported MSC circulation phenomena. Deep-femoral vein (FV) and matched peripheral vein blood samples (PVBs) were collected from patients undergoing lower-limb orthopaedic procedures during surgery (tibia using conventional sequential reaming, n = 9, femur using reamer/irrigator/aspirator (RIA), n = 15). PVBs were also taken from early (n = 15) and established (n = 12) rheumatoid arthritis (RA) patients and healthy donors (n = 12). Colony-forming unit-fibroblasts (CFU-Fs) were found in 17/36 FVBs but only 7/74 PVBs (mostly from femoral RIA); highly proliferative clonogenic cells were not generated. Only one colony was found in control/RA samples (n = 28). The rare CFU-Fs’ MSC nature was confirmed by phenotypic: CD105+/CD73+/CD90+ and CD19−/CD31−/CD33−/CD34−/CD45−/CD61−, and molecular profiles with 39/80 genes (including osteo-, chondro-, adipo-genic and immaturity markers) similar across multiple MSC tissue controls, but not dermal fibroblasts. Analysis of FVB-MSCs suggested that their likely origin was bone marrow as only two differences were observed between FVB-MSCs and IC-BM-MSCs (ACVR2A, p = 0.032 and MSX1, p = 0.003). Stromal cells with the phenotype and molecular profile of MSCs were scarcely found in the circulation, supporting the hypothesis that their very rare presence is likely linked to biophysical micro-damage caused by skeletal trauma (here orthopaedic manipulation) rather than specific molecular cues to a circulatory pool of MSCs capable of repair of remote organs or tissues. These findings support the use of organ resident cells or MSCs placed in situ to repair tissues rather than systemic administration.

A model study for the manufacture and validation of clinical-grade deciduous dental pulp stem cells for chronic liver fibrosis treatment

BackgroundHuman deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Recent studies demonstrated that hDPSCs exhibited in vivo anti-fibrotic and anti-inflammatory action and in vivo hepatogenic-associated liver regeneration, suggesting that hDPSCs may offer a promising source with great clinical demand for treating liver diseases. However, how to manufacture ex vivo large-scale clinical-grade hDPSCs with the appropriate quality, safety, and preclinical efficacy assurances remains unclear.MethodsWe isolated hDPSCs from human deciduous dental pulp tissues formed by the colony-forming unit-fibroblast (CFU-F) method and expanded them under a xenogeneic-free and serum-free (XF/SF) condition; hDPSC products were subsequently stored by two-step banking including a master cell bank (MCB) and a working cell bank (WCB). The final products were directly thawed hDPSCs from the WCB. We tested the safety and quality check, stem cell properties, and preclinical potentials of final hDPSC products and hDPSC products in the MCB and WCB.ResultsWe optimized manufacturing procedures to isolate and expand hDPSC products under a XF/SF culture condition and established the MCB and the WCB. The final hDPSC products and hDPSC products in the MCB and WCB were validated the safety and quality including population doubling ability, chromosome stability, microorganism safety, and stem cell properties including morphology, cell surface marker expression, and multipotency. We also evaluated the in vivo immunogenicity and tumorigenicity and validated in vivo therapeutic efficacy for liver regeneration in a CCl4-induced chronic liver fibrosis mouse model in the final hDPSC products and hDPSC products in the WCB.ConclusionThe manufacture and quality control results indicated that the present procedure could produce sufficient numbers of clinical-grade hDPSC products from a tiny deciduous dental pulp tissue to enhance clinical application of hDPSC products in chronic liver fibrosis.

Characteristics of Bone Marrow Progenitor Cells of C57BL/6N Mice after 30-Day Hindlimb Suspension and 12-Hour Readaptation to Support Loading

The functional status of bone marrow (BM) progenitor cells, which provide hematopoiesis and homeostasis of bone tissue, determines many processes which take a place in spaceflight (SF) and subsequent recovery to gravity. It is known that SF factors and simulation of their effects do irreversible damage to the pool of hematopoietic and stromal progenitor cells. Meanwhile, changes in the functional activity of BM progenitor cells after simulation of SF effects were more pronounced than in the case of a real SF. The aim of the study was to examine processes of recovery of the functional activity of BM progenitor cells during readaptaion to loading after 30-day hindlimb suspension. Studies were performed on three groups of male mice: vivarium control, suspension, and readaptation. The following characteristics were monitored: number of nucleated BM cells, immunophenotype, proliferative activity of stromal BM cells, number of hematopoietic colony-forming units (CFU), CFUs of fibroblasts, and differentiating capabilities of BM hematopoietic and stromal precursors. Hindlimb suspension resulted in decreased number of fibroblast and hematopoietic CFU, inhibition of spontaneous osteodifferentiation of stromal progenitors and decreased expression of stromal markers. The readaptaion period was accompanied with restoration of the functional activity of stromal progenitors, while complete recovery of hematopoiesis was not observed.

The Phenotype and Functional Activity of Mesenchymal Stromal Cells in Pediatric Patients with Non-Malignant Hematological Diseases

As the biology of mesenchymal stromal cells (MSCs) in patients with non-malignant hematological diseases (NMHD) is poorly understood, in the current study we performed a basic characterization of the phenotype and functional activity of NMHD-MSCs. Bone marrow (BM) of patients with thalassemia major (TM) possessed a significantly higher number of nucleated cells (BM-MNCs)/mL BM than healthy donors (P < 0.0001), which however did not result in a higher number of colony forming units-fibroblast (CFU-F) per milliliter BM. In contrast, from 1 × 106 BM-MNCs of patients with sickle cell disease (SCD) were generated significantly more CFU-Fs than from TM-BM-MNCs (P < 0.013) and control group (P < 0.02). In addition, NMHD-MSCs expressed significantly lower levels of CD146 molecule, demonstrated an equal proliferation potential and differentiated along three lineages (osteoblasts, chondrocytes and adipocytes) as healthy donors’ MSCs, with exception of TM-MSCs which differentiated weakly in adipocytes. In contrast to other NMHD-MSCs and healthy donors’ MSCs, TM-MSCs demonstrated an impaired in vitro immunosuppressive potential, either. Noteworthy, the majority of the immunosuppressive effect of NMHD-MSCs was mediated through prostaglandin-E2 (PGE2), because indomethacin (an inhibitor of PGE2 synthesis) was able to significantly reverse this effect. Our results indicate therefore that NMHD-MSCs, except TM-MSCs, may be used as an autologous cell-based therapy for post-transplant complications such as graft failure, graft-versus-host disease (GvHD) and osteonecrosis.

Safety and Efficacy of Human Adipose-Derived Stromal/Stem Cell Therapy in an Immunocompetent Murine Pressure Ulcer Model

Pressure injuries/ulcers are frequent complications in elderly, paraplegic, and quadriplegic patients, which account for considerable cost to the international health care economy and remain refractory to current treatment options. Autologous or allogeneic adult stromal/stem cells represent an alternative therapeutic approach. The current study extends prior findings by exploring the safety and efficacy of human adipose-derived stromal/stem cell (ASC) therapy in an established immunocompetent murine skin pressure ulcer model where dermal fibroblast cells (DFCs) served as a control. Human adipose tissue was processed using a closed system device designed for point-of-care use in the operating room and on file with the Food and Drug Administration. Cell characterization was performed using colony-forming unit-fibroblast, differentiation, and immunophenotypic assays in vitro. Wound healing was assessed over a 20-day period based on photomicrographs, histology, and immunohistochemistry. The isolated human ASCs displayed significantly greater colony formation relative to DFCs while both populations exhibited comparable immunophenotype and differentiation potential. Both fresh and cryopreserved human ASCs significantly accelerated and enhanced wound healing in young (2 month) mice of both sexes relative to DFC controls based on tissue architecture and CD68+ cell infiltration. In contrast, while injection of either fresh or cryopreserved human ASCs was safe in older mice, the fresh ASCs significantly enhanced wound closure relative to the cryopreserved ASCs. Overall, these findings support the safety and efficacy of human ASCs isolated using a closed system device designed for clinical procedures in the future treatment of pressure injuries.

Evaluation of the Consistency and Composition of Commercially Available Bone Marrow Aspirate Concentrate Systems.

Background:Bone marrow aspirate (BMA) concentrate (BMAC) has gained popularity as a treatment modality for various orthopaedic conditions; however, there are still inconsistencies in its reported therapeutic efficacy. This may be because of the many different commercial BMAC preparation systems used clinically, which generate dissimilar concentrate products.Purpose:To compare 3 commercially available BMAC preparation systems: Harvest SmartPrep 3, Biomet BioCUE, and Arthrex Angel. We evaluated the consistency of each of these systems and compared the composition of their concentrate products.Study Design:Controlled laboratory study.Methods:A total of 10 patients donated whole blood and BMA, which were combined and processed with the 3 different BMAC preparation systems. Samples were taken before and after processing for the measurement of white blood cells (WBC), platelets (PLT), CD34+ cells, and colony-forming unit–fibroblast (CFU-F). To evaluate consistency, the variances of cell yield and concentration increase from baseline for each cell type were compared between systems. To compare concentrate product composition, differences between the systems’ mean cell yield and concentration increase from baseline for each cell type were evaluated.Results:The Harvest system (variance, 0.25) concentrated WBC more consistently than the Arthrex system (variance, 3.25) (P = .024), but no other differences in consistency were noted between the 3 systems. The Harvest system recovered the greatest percentage of CFU-F (82.4% ± 18.2%), CD34+ cells (81.1% ± 28.5%), and WBC (77.3% ± 8.6%), whereas the Biomet system recovered the greatest percentage of PLTs (92.9% ± 27.3%). The Arthrex system concentrated PLT to the greatest degree (11.10 ± 2.05 times baseline), while the Biomet system concentrated WBC to the greatest degree (5.99 ± 1.04 times baseline).Conclusion:The consistency of the 3 systems was similar for all but 1 of the evaluated cell types. However, the composition of the concentrate products differed across systems. This may grant each system unique advantages without having to sacrifice reproducibility.Clinical Relevance:Understanding the consistency of different BMAC preparation systems and their product makeup may aid in determining optimal therapeutic doses of different cell types.

EFFICIENCY OF BONE MARROW PRECURSOR CELL COLONY-FORMING AS A PREDICTOR OF DISEASE COURSE IN PLASMA CELL MYELOMA PATIENTS WITH A HISTORY OF RADIATION EXPOSURE.

Assessment of role of the bone marrow colony-forming efficiency in plasma cell myeloma patients at different stages of treatment as a prognostic criterion for the disease course. The colony forming efficiency (CFE) was assayed in stage I-II plasma cell myeloma (PCM)patients (n = 37) aged 42-73, namely in patients survived after the Chornobyl NPP accident (n = 21) and persons notexposed to ionizing radiation (n = 16). There were 11 males exposed to ionizing radiation and having got stage I PCM,9 males and 3 females exposed and having got stage II PCM, 3 males and 3 females not exposed and having got stageI PCM, 6 males and 2 females not exposed and having got stage II PCM. Healthy persons (n = 20) were included in thecontrol group. Number of the bone marrow (BM) granulocyte-macrophage colony-forming units (CFU-GM) in both exposedand not exposed PCM patients depended on a disease stage. CFU-GM was (16.7 ± 1.2) in the stage I PCM patients vs.(11.1 ± 1.1) in the stage II PCM ones both being lower (p < 0.05) compared to control (64.5 ± 2.2). Changes in cluster formation were similar, i.e. (37.7 ± 1.6) and (19.4 ± 1.3) correspondingly in the stage I and stage II PCM patients.Respective values in control were (89.8 ± 3.6). The CFE in stage I and stage II PCM patients at the time of diagnosiswas lower (5.7 ± 1.5 and 2.4 ± 1.1 respectively) vs. control (39.5 ± 1.51, p < 0.05), but has increased in remission upto (29. 6 ± 1.8) and (13.8 ± 1.2) respectively. There was no difference at that between the irradiated and non-irradiated patients. Number of the fibroblast colony-forming units (CFU-F) in the stage I and stage II PCM patients duringdiagnosis, namely (43.9 ± 5.4) and (22.5 ± 3.7), was lower (p < 0.05) vs. control (110.5 ± 4.9). Upon reaching remission the CFU-F value increased significantly (p < 0.05), reaching (87.4 ± 4.2) and (55.6 ± 2.7) correspondingly in thestage I and stage II PCM patients. Dependence of the BM cell CFE on the stage of PCM and presence or absence of remission was established. Prognostic value of the CFE of BM CFU-GM in terms of life span of patients was shown (Ro Spearm = 0.39,p < 0.02), namely in case of CFE > 20 before the polychemotherapy administration the life span of PCM patients wassignificantly longer vs. cases of CFE < 20.

Reliable isolation of human mesenchymal stromal cells from bone marrow biopsy specimens in patients after allogeneic hematopoietic cell transplantation

Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit–fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 106 total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 106; range, 0–2.3 × 107 P0 MSCs versus 5.4 × 104; range, 0–8.9 × 106; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions.