The stem cell revolution: on the role of CD164 as a human stem cell marker.

Accurately defining hierarchical relationships between human stem cells and their progeny, and using this knowledge for new cellular therapies, will undoubtedly lead to further successful treatments for life threatening and chronic diseases, which represent substantial burdens on patient quality of life and to healthcare systems globally. Clinical translation relies in part on appropriate biomarker, in vitro manipulation and transplantation strategies. CD164 has recently been cited as an important biomarker for enriching both human haematopoietic and skeletal stem cells, yet a thorough description of extant human CD164 monoclonal antibody (Mab) characteristics, which are critical for identifying and purifying these stem cells, was not discussed in these articles. Here, we highlight earlier but crucial research describing these relevant characteristics, including the differing human CD164 Mab avidities and their binding sites on the human CD164 sialomucin, which importantly may affect subsequent stem cell function and fate.

Correction: A human bone marrow mesodermal-derived cell population with hemogenic potential.

At the time of publication the funding information was omitted from the article - this has now been corrected in both the HTML and the PDF.

A human bone marrow mesodermal-derived cell population with hemogenic potential.

The presence, within the human bone marrow, of cells with both endothelial and hemogenic potential has been controversial. Herein, we identify, within the human fetal bone marrow, prior to establishment of hematopoiesis, a unique APLNR+, Stro-1+ cell population, co-expressing markers of early mesodermal precursors and/or hemogenic endothelium. In adult marrow, cells expressing similar markers are also found, but at very low frequency. These adult-derived cells can be extensively culture expanded in vitro without loss of potential, they preserve a biased hemogenic transcriptional profile, and, upon in vitro induction with OCT4, assume a hematopoietic phenotype. In vivo, these cells, upon transplantation into a fetal microenvironment, contribute to the vasculature, and generate hematopoietic cells that provide multilineage repopulation upon serial transplantation. The identification of this human somatic cell population provides novel insights into human ontogenetic hematovascular potential, which could lead to a better understanding of, and new target therapies for, malignant and nonmalignant hematologic disorders.

Multiple Drug-Toxicity Involving Novel Psychoactive Substances, 3-Fluorophenmetrazine and U-47700.

3-Fluorophenmetrazine (3-FPM) is a stimulant-like novel psychoactive substance (NPS) and fluorinated analog of phenmetrazine that has recently appeared on the recreational drug market, with limited published information. Likewise, the synthetic opioid U-47700 has gained popularity among recreational drug users and is frequently detected in postmortem casework. We present the case history, autopsy and toxicological findings of a fatality involving the designer drugs 3-FPM and U-47700 for the first time in the literature. A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of 3-FPM in whole blood, with a 0.001-0.100 mg/L analytical range. The method met the requirements for acceptable linearity, bias and precision. 3-FPM was detected along with U-47700 and other drugs including amitriptyline, nortriptyline, methamphetamine, amphetamine, diazepam, nordiazepam, temazepam, and the designer benzodiazepines flubromazolam and delorazepam. 3-FPM was quantified in the decedent's peripheral (femoral) and central (aortic) blood at 2.4 and 2.6 mg/L, respectively. These concentrations are similar to reported concentrations in non-fatal intoxications. U-47700 was present in peripheral blood at a semi-quantitative concentration of 0.36 mg/L, consistent with reported U-47700 postmortem concentrations. The cause of death was considered multiple drug-toxicity (3-FPM, U-47700, amitriptyline, methamphetamine, diazepam, temazepam, flubromazolam and delorazepam) and the manner of death ruled an accident. This case illustrates the dangers of polysubstance use and discusses the potential overlap between recreational and fatal concentrations for some NPS.

Non-fucosylated CB CD34+ cells represent a good target for enforced fucosylation to improve engraftment following cord blood transplantation.

BACKGROUND AIMS: Despite ethnic diversity and ready availability of cryopreserved, human leukocyte antigen-typed cord blood (CB), delayed engraftment remains a significant hurdle to successful CB transplantation. Suboptimal homing of CB hematopoietic stem and progenitor cells (HSPCs) to the hematopoietic microenvironment (HM) is thought to be responsible and due to low levels of HSPC fucosylation. Fucosylation (decoration with sialyl-LewisX) may improve HSPC homing to HM by increasing the strength of HSPC/E-selectin interactions, where E-selectin is constitutively expressed by HM microvasculature. Enforced fucosylation of CB HSPCs using fucosyltransferases, increases the rate and magnitude of engraftment in xenogeneic transplant models. However, it is unclear whether endogenously fucosylated and non-fucosylated CB HSPC are qualitatively identical or whether endogenous fucosylation marks a qualitative difference between CB HSPC. If qualitatively identical, non-fucosylated CB HSPCs represent a good target for enforced fucosylation with improved engraftment conferred on an increased number of otherwise qualitatively identical HSPC. If qualitatively different, then conferring engraftment upon a majority, possibly lower "quality," non-fucosylated HSPCs by enforced fucosylation might inadvertently compromise engraftment. METHODS: Functional (xenogeneic engraftment, colony-forming unit and selectin-binding assays) and phenotypic analyses of fluorescence-activated cell sorting-isolated, endogenously fucosylated and non-fucosylated CB CD34+ cells were performed. RESULTS: Endogenous fucosylation of CB HSPCs exists as a continuum. Endogenously fucosylated HSPCs engrafted more efficiently in a xenogeneic transplantation model than non-fucosylated HSPCs. Outside of the differences in endogenous fucosylation, no other qualitative (functional and/or phenotypic) differences were identified. DISCUSSION: The majority of endogenously non-fucosylated CB HSPCs represent a good target for enforced fucosylation with the goal of improving engraftment following CB transplantation.

A unique microenvironment in the developing liver supports the expansion of megakaryocyte progenitors.

The fetal liver is the site of a major expansion of the hematopoietic stem cell (HSC) pool and is also a privileged organ to study megakaryocyte progenitor differentiation. We identified in the mouse fetal liver at day 13.5 a discrete stromal cell population harboring a CD45-TER119-CD31-CD51+VCAM-1+PDGFRα- (V+P-) phenotype that lacked colony-forming unit fibroblast activity and harbored an hepatocyte progenitor signature. This previously undescribed V+P- population efficiently supported megakaryocyte production from mouse bone marrow HSC and human peripheral blood HSC-myeloid progenitors cultured in the presence of limited cytokine concentrations. Megakaryocytes obtained in V+P- cocultures were polyploid, positive for CD41/CD42c, and efficiently produced proplatelets. Megakaryocyte production appeared to be mediated by an expansion of the progenitor compartment through HSC-stromal cell contact. In conclusion, the fetal liver contains a unique cellular microenvironment that could represent a platform for the discovery of regulators of megakaryopoiesis.

Mesenchymal stem cells from cortical bone demonstrate increased clonal incidence, potency, and developmental capacity compared to their bone marrow-derived counterparts.

In this study, we show that matrix dense cortical bone is the more potent compartment of bone than bone marrow as a stromal source for mesenchymal stem cells as isolated from adult rats. Lineage-depleted cortical bone-mesenchymal stem cells demonstrated >150-fold enrichment of colony forming unit-fibroblasts per cell incidence. compared to lineage-depleted bone marrow-mesenchymal stem cells, corresponding to a 70-fold increase in absolute recovered colony forming unit-fibroblasts. The composite phenotype Lin(-)/CD45(-)/CD31(-)/VLA-1(+)/Thy-1(+) enriched for clonogenic mesenchymal stem cells solely from cortical bone-derived cells from which 70% of clones spontaneously differentiated into all lineages of bone, cartilage, and adipose. Both populations generated vascularized bone tissue within subcutaneous implanted collagen scaffolds; however, cortical bone-derived cells formed significantly more osteoid than bone marrow counterparts, quantified by histology. The data demonstrate that our isolation protocol identifies and validates mesenchymal stem cells with superior clonal, proliferative, and developmental potential from cortical bone compared to the bone marrow niche although marrow persists as the typical source for mesenchymal stem cells both in the literature and current pre-clinical therapies.

Frequency and Determinants of Preventive Care Counseling by HIV Medical Care Providers during Encounters with Newly Diagnosed and Established HIV-Infected Patients.

This study evaluates the frequency and determinants of preventive care counseling by HIV medical care providers (HMCPs) during encounters with newly diagnosed and established HIV-infected patients. Data used were from a probability sample of HMCPs in Houston/Harris County, Texas, surveyed in 2009. Overall, HMCPs offered more preventive care counseling to newly diagnosed than the established patients (adjusted odds ratio [AOR] = 7.28; 95% confidence interval [CI] = 2.86-16.80). They were more likely to counsel newly diagnosed patients than the established ones on medication and adherence (AOR = 14.70; 95% CI = 1.24-24.94), HIV risk reduction (AOR = 5.91; 95% CI = 0.48-7.13), and disease screening (AOR = 7.20; 95% CI = 0.72-11.81). HIV medical care providers who were less than 45 years of age, infectious disease specialists, and had less than 30 minutes of encounter time were less likely to counsel patients regardless of the status. Our findings suggest the need for HMCPs to improve their preventive care counseling efforts, in order to help patients build skills for adopting and maintaining safe behavior that could assist in reducing the risk of HIV transmission.

Enforced fucosylation of cord blood hematopoietic cells accelerates neutrophil and platelet engraftment after transplantation.

Delayed engraftment is a major limitation of cord blood transplantation (CBT), due in part to a defect in the cord blood (CB) cells' ability to home to the bone marrow. Because this defect appears related to low levels of fucosylation of cell surface molecules that are responsible for binding to P- and E-selectins constitutively expressed by the marrow microvasculature, and thus for marrow homing, we conducted a first-in-humans clinical trial to correct this deficiency. Patients with high-risk hematologic malignancies received myeloablative therapy followed by transplantation with 2 CB units, one of which was treated ex vivo for 30 minutes with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose to enhance the interaction of CD34(+) stem and early progenitor cells with microvessels. The results of enforced fucosylation for 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. The median time to neutrophil engraftment was 17 days (range, 12-34 days) compared with 26 days (range, 11-48 days) for controls (P = .0023). Platelet engraftment was also improved: median was 35 days (range, 18-100 days) compared with 45 days (range, 27-120 days) for controls (P = .0520). These findings support ex vivo fucosylation of multipotent CD34(+) CB cells as a clinically feasible means to improve engraftment efficiency in the double CBT setting. The trial is registered to www.clinicaltrials.gov as #NCT01471067.

Expression of a surface antigen (MA6) by peripheral blood CD34+ cells is correlated with improved platelet engraftment and may explain delayed platelet engraftment following cord blood transplantation.

CD34(+) cell dose provides a measure of hematopoietic tissue that predicts the rate of engraftment upon transplant. It is positively correlated with multiple measures of hematopoietic recovery, including platelet engraftment. Here we identify a subpopulation of CD34(+) cells that coexpress a surface antigen--MA6, which is more positively correlated with platelet engraftment in a clinical setting than CD34(+) alone. The specific identity and function of MA6 remain to be determined, however, it is expressed by primitive megakaryocyte (MK) progenitors, but is lost with differentiation and is not expressed by platelets. Commitment of CD34(+)MA6(+) cells to the MK lineage was confirmed by in vitro assays and their significance in hematopoietic transplantation explored by flow cytometric analysis of cryopreserved samples of granulocyte colony stimulating factor-mobilized peripheral blood progenitor cell (PBPC) products along with a retrospective analysis of platelet engraftment data. Platelet engraftment by day 21 was predicted by receipt of ≥ 6 × 10(6) CD34(+) cells/kg or ≥ 0.3 × 10(6) CD34(+)MA6(+) cells/kg. Subsequent analysis of cord blood (CB) CD34(+) cells revealed <0.2% coexpressed MA6(+), compared to 8% of PBPC CD34(+) cells. This low proportion of CD34(+)MA6(+) cells may be responsible, at least in part, for the delayed platelet engraftment associated with CB transplantation. However, platelet engraftment is markedly improved in recipients of ex vivo-expanded CB. This may be a consequence of an increased proportion of CD34(+)MA6(+) cells present in the ex vivo-expanded product and also suggests that optimizing ex vivo culture conditions to generate CD34(+)MA6(+) cells might further improve platelet engraftment in CB recipients.

Manufacturing challenges in regenerative medicine.

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment.

BACKGROUND AIMS: Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo. METHODS: CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment. RESULTS: Both FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes. CONCLUSIONS: Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.

A comparison of the behavioral and anatomical outcomes in sub-acute and chronic spinal cord injury models following treatment with human mesenchymal precursor cell transplantation and recombinant decorin.

This study assessed the potential of highly purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) in combination with the anti-scarring protein decorin to repair the injured spinal cord (SC). Donor hMPCs isolated from spinal cord injury (SCI) patients were transplanted into athymic rats as a suspension graft, alone or after previous treatment with, core (decorin(core)) and proteoglycan (decorin(pro)) isoforms of purified human recombinant decorin. Decorin was delivered via mini-osmotic pumps for 14 days following sub-acute (7 day) or chronic (1 month) SCI. hMPCs were delivered to the spinal cord at 3 weeks or 6 weeks after the initial injury at T9 level. Behavioral and anatomical analysis in this study showed statistically significant improvement in functional recovery, tissue sparing and cyst volume reduction following hMPC therapy. The combination of decorin infusion followed by hMPC therapy did not improve these measured outcomes over the use of cell therapy alone, in either sub-acute or chronic SCI regimes. However, decorin infusion did improve tissue sparing, reduce spinal tissue cavitation and increase transplanted cell survivability as compared to controls. Immunohistochemical analysis of spinal cord sections revealed differences in glial, neuronal and extracellular matrix molecule expression within each experimental group. hMPC transplanted spinal cords showed the increased presence of serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted hMPCs for up to 2 months; however, no evidence of hMPC transdifferentiation into neuronal or glial phenotypes. The number of hMPCs was dramatically reduced overall, and no transplanted cells were detected at 8 weeks post-injection using lentiviral GFP labeling and human nuclear antigen antibody labeling. The presence of recombinant decorin in the cell transplantation regimes delayed in part the loss of donor cells, with small numbers remaining at 2 months after transplantation. In vitro co-culture experiments with embryonic dorsal root ganglion explants revealed the growth promoting properties of hMPCs. Decorin did not increase axonal outgrowth from that achieved by hMPCs. We provide evidence for the first time that (Stro-1(+)) hMPCs provide: i) an advantageous source of allografts for stem cell transplantation for sub-acute and chronic spinal cord therapy, and (ii) a positive host microenvironment that promotes tissue sparing/repair that subsequently improves behavioral outcomes after SCI. This was not measurably improved by recombinant decorin treatment, but does provide important information for the future development and potential use of decorin in contusive SCI therapy.

Heterogeneity and immunophenotypic plasticity of malignant cells in human liposarcomas.

Liposarcomas are tumors arising in white adipose tissue (WAT) with avidity for local recurrence. Aggressive dedifferentiated liposarcomas (DDLS) may arise from well-differentiated subtypes (WDLS) upon disease progression, however, this key issue is unresolved due in large part to knowledge gaps about liposarcoma cellular composition. Here, we wished to improve insights into liposarcoma cellular hierarchy. Tumor section analysis indicated that the populations, distinguishable based on the expression of CD34 (a marker of adipocyte progenitors) and CD36 (a marker of adipocyte differentiation), occupy distinct intra-tumoral locations in both WDLS and DDLS. Taking advantage of these markers, we separated cells from a panel of fresh human surgical specimens by fluorescence-activated cell sorting (FACS). Based on chromosome analysis and the culture phenotypes of the composing populations, we demonstrate that malignant cells comprise four mesenchymal populations distinguished by the expression of CD34 and CD36, while vascular (CD31+) and hematopoietic (CD45+) components are non-neoplastic. Finally, we show that mouse xenografts are derivable from both CD36-negative and CD36-positive DDLS cells, and that each population recreates the heterogeneity of CD36 expression in vivo. Combined, our results show that malignant cells in WDLS and DDLS can be classified according to distinct stages of adipogenesis and indicate immunophenotypic plasticity of malignant liposarcoma cells.

Multiple role relationships in healthcare education.

Healthcare training environments, particularly in multidisciplinary training settings, present unique ethical dilemmas as a result of the multiple relationships faculty must balance while working with trainees. The historical and current perspectives on multiple roles in training environments will first be summarized. Evidence of a gap between the extant discipline specific guidelines and the realities of situations that occur in healthcare training will then be revealed, as illustrated in a case example. Primary care medicine training environments are highly nuanced, potentially leading to an infinite number of ambiguous situations that require a generalizable model for managing multiple roles. Rather than recommend specific modifications to existing ethical guidelines, a new model emphasizing role awareness and decision making when challenges in healthcare training settings arise is proposed. Recommendations for the case example using the model are offered. All professionals are prone to boundary transgressions; explicit training about and the maintenance of appropriate role balance will help to ensure high-functioning relationships and maximize the quality of patient care, resident education, faculty and resident satisfaction, and modeling of professional behavior to improve competencies as clinicians and educators.

The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine.

Mesenchymal stem cells (MSCs) are the focus of intensive efforts worldwide directed not only at elucidating their nature and unique properties but also developing cell-based therapies for a diverse range of diseases. More than three decades have passed since the original formulation of the concept, revolutionary at the time, that multiple connective tissues could emanate from a common progenitor or stem cell retained in the postnatal bone marrow. Despite the many important advances made since that time, substantial ambiguities still plague the field regarding the nature, identity, function, mode of isolation and experimental handling of MSCs. These uncertainties have a major impact on their envisioned therapeutic use.

Human mesenchymal precursor cells (Stro-1⁺) from spinal cord injury patients improve functional recovery and tissue sparing in an acute spinal cord injury rat model.

This study aimed to determine the potential of purified (Stro-1(+)) human mesenchymal precursor cells (hMPCs) to repair the injured spinal cord (SC) after transplantation into T-cell-deficient athymic RNU nude rats following acute moderate contusive spinal cord injury (SCI). hMPCs were isolated from the bone marrow (BM) stroma of SCI patients and transplanted as a suspension graft in medium [with or without immunosuppression using cyclosporin A (CsA)]. Extensive anatomical analysis shows statistically significant improvement in functional recovery, tissue sparing, and cyst reduction. We provide quantitative assessment of supraspinal projections in combination with functional outcomes. hMPC-transplanted animals consistently achieved mean BBB scores of 15 at 8 weeks post injury. Quantitative histological staining revealed that graft-recipient animals possessed more intact spinal tissue and reduced cyst formation than controls. Fluorogold (FG) retrograde tracing revealed sparing/regeneration of supraspinal and local propriospinal axonal pathways, but no statistical differences were observed compared to controls. Immunohistochemical analysis revealed increased serotonergic (5-HT) and sensory (CGRP) axonal growth within and surrounding transplanted donor hMPCs 2 weeks posttransplantation, but no evidence of hMPC transdifferentiation was seen. Although hMPCs initially survive at 2 weeks posttransplantation, their numbers were dramatically reduced and no cells were detected at 8 weeks posttransplantation using retroviral/lentiviral GFP labeling and a human nuclear antigen (HNA) antibody. Additional immunosuppression with CsA did not improve hMPC survival or their ability to promote tissue sparing or functional recovery. We propose Stro-1(+)-selected hMPCs provide (i) a reproducible source for stem cell transplantation for SC therapy and (ii) a positive host microenvironment resulting in the promotion of tissue sparing/repair that subsequently improves behavioral outcomes after SCI. Our results provide a new candidate for consideration as a stem cell therapy for the repair of traumatic CNS injury.

Cord-blood engraftment with ex vivo mesenchymal-cell coculture.

BACKGROUND: Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. METHODS: We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. RESULTS: Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram--doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). CONCLUSIONS: Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316.).

Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells.

Directed specification and prospective isolation of chondrogenic paraxial mesoderm progeny from human pluripotent stem (PS) cells have not yet been achieved. Here we report the successful generation of KDR(-)PDGFRα(+) progeny expressing paraxial mesoderm genes and the mesendoderm reporter MIXL1-GFP in a chemically defined medium containing the canonical WNT signaling activator, BMP-inhibitor, and the Nodal/Activin/TGFß signaling controller. Isolated (GFP(+))KDR(-)PDGFRα(+) mesoderm cells were sensitive to sequential addition of the three chondrogenic factors PDGF, TGFß and BMP. Under these conditions, the cells showed robust chondrogenic activity in micromass culture, and generated a hyaline-like translucent cartilage particle in serum-free medium. In contrast, both STRO1(+) mesenchymal stem/stromal cells from adult human marrow and mesenchymal cells spontaneously arising from hPS cells showed a relatively weaker chondrogenic response in vitro, and formed more of the fibrotic cartilage particles. Thus, hPS cell-derived KDR(-)PDGFRα(+ )paraxial mesoderm-like cells have potential in engineered cartilage formation and cartilage repair.