Fibroblasts and Mesenchymal Stromal/Stem Cells Are Phenotypically Indistinguishable.

BACKGROUND/AIMS: Human mesenchymal stromal/stem cells (MSCs), derived from many different tissues, are characterized by a fibroblast-like morphology, the expression of certain cell surface markers and their ability to differentiate into adipocytes, chondrocytes and osteoblasts. A number of studies have shown that MSCs share many characteristics with fibroblasts; however, there is no well-defined set of phenotypic characteristics that could distinguish between these 2 types of cells. METHODS: We used 4 well-established human fibroblast strains from 3 different tissue sources and several human MSC strains from 2 different tissue sources to compare the phenotypic and immunological characteristics of these cells. RESULTS: Fibroblast strains had a similar morphology to MSCs, expressed the same cell surface markers as MSCs and could also differentiate into adipocytes, chondrocytes and osteoblasts. Also, similar to MSCs, these fibroblasts were capable of suppressing T cell proliferation and modulating the immunophenotype of macrophages. We also show that MSCs deposit extracellular matrices of collagen type I and fibronectin, and express FSP1 in patterns similar to fibroblasts. CONCLUSIONS: Based on currently accepted definitions for cultured human MSCs and fibroblasts, we could not find any immunophenotypic property that could make a characteristic distinction between MSCs and fibroblasts.

Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model.

BACKGROUND AIMS: Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. METHODS: We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4(+) fraction of CD34(+) HSCs, we could improve engraftment. Human cord blood-derived CD34(+) cells and human bone marrow-derived MSCs were used for these studies. RESULTS: When MSCs were transplanted 1 week before CD34(+) cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34(+) cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4(+) cells in the CD34(+) population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. CONCLUSIONS: Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4-SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.

Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model.

Until now, ex vivo generation of CD34(+) hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14(+) monocyte-derived macrophages to generate CD34(+) cells from hESCs in vitro. Human ESC-derived CD34(+) cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34(+) cells. Finally, transplantation of purified hESC-derived CD34(+) cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This in vivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34(+) cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.

In vivo generation of beta-cell-like cells from CD34(+) cells differentiated from human embryonic stem cells.

OBJECTIVE: CD34(+) cells, present within the bone marrow, have previously been shown to possess pancreatic endocrine potential. Based on this observation, we explored the capacity of CD34(+) cells derived in culture from the differentiation of human embryonic stem cells (hESC), for their in vivo pancreatic endocrine capacity. MATERIALS AND METHODS: Sheep were transplanted with hESC-derived CD34(+) cells, as well as nonsorted differentiated cultures. Transplantations were carried out with in utero intraperitoneal injections prior to development of the immune system in the fetus so that tolerance toward foreign antigens was acquired during gestation and persisted in the adult. RESULTS: All cell populations that were tested demonstrated human cellular activity and long-term presence up to 5 years. However, the in vivo beta-cell-like activity achieved from the transplantation of the sorted CD34(+) cell population was not augmented by transplanting the entire cell population from which the CD34(+) cells were isolated. Human DNA and insulin messenger RNA were detected in sheep pancreases. An average of 1.51 ng/mL human C-peptide was detected in serum from eight animals transplanted with differentiated cell populations and assayed up to 55 months posttransplantation. Transplantation of as few as 23,500 cells resulted in long-term sustainable beta-cell-like activity. Teratomas were absent in the transplanted animals. CONCLUSION: Our data suggest that hESC-derived CD34(+) cells have a potential for long-term in vivo endocrine cellular activity that could prove useful in regenerative medicine. Because the same cell population has previously been shown to contain hematopoietic potential, it could be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes.

Persistent circulating human insulin in sheep transplanted in utero with human mesenchymal stem cells.

OBJECTIVE: To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero. MATERIALS AND METHODS: A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver, and bone marrow. RESULTS: Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79%, while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well. CONCLUSION: We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow-derived MSC. In vivo results suggest the pMSC engraft, differentiate, and secrete human insulin from the sheep pancreas.

Immunologic recovery following autologous stem-cell transplantation with pre- and posttransplantation rituximab for low-grade or mantle cell lymphoma.

BACKGROUND: Rituximab may improve transplant outcomes but may delay immunologic recovery. PATIENTS AND METHODS: Seventy-seven patients with low-grade or mantle cell lymphoma received autologous stem-cell transplantation (ASCT) on a phase II study. Rituximab 375 mg/m(2) was administered 3 days before mobilization-dose cyclophosphamide, then weekly for four doses after count recovery from ASCT. Immune reconstitution was assessed. RESULTS: Sixty percent of transplants occurred in first remission. Actuarial event-free survival (EFS) and overall survival (OS) were 60% and 73%, respectively, at 5 years, with 7.2-year median follow-up for OS in surviving patients. Median EFS was 8.3 years. Older age and transformed lymphomas were independently associated with inferior EFS, whereas day 60 lymphocyte counts did not predict EFS or late infections. Early and late transplant-related mortality was 1% and 8%, with secondary leukemia in two patients. B-cell counts recovered by 1-2 years; however, the median IgG level remained low at 2 years. Late-onset idiopathic neutropenia, generally inconsequential, was noted in 43%. CONCLUSION: ASCT with rituximab can produce durable remissions on follow-up out to 10 years. Major infections do not appear to be significantly increased or to be predicted by immune monitoring.

Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity.

PURPOSE: Rituximab has been reported to have little activity in small lymphocytic lymphoma (SLL)/chronic lymphocytic leukemia (CLL) and to be associated with significant infusion-related toxicity. This study sought to decrease the initial toxicity and optimize the pharmacokinetics with an alternative treatment schedule. PATIENTS AND METHODS: Thirty three patients with SLL/CLL received dose 1 of rituximab (100 mg) over 4 hours. In cohort I (n = 3; 250 mg/m(2)) and cohort II (n = 7; 375 mg/m(2)) rituximab was administered on day 3 and thereafter three times weekly for 4 weeks using a standard administration schedule. Cohort III (n = 23; 375 mg/m(2)) administered rituximab similar to cohort II for the first two treatments and then over 1 hour thereafter. RESULTS: A total of 33 CLL/SLL patients were enrolled; only one patient discontinued therapy because of infusion-related toxicity. Thirteen patients developed transient hypoxemia, hypotension, or dyspnea that were associated with significant changes in baseline interleukin-6, interleukin-8, tumor necrosis factor alpha, and interferon gamma compared with those not experiencing such reactions. Infusion-related toxicity occurred more commonly in older (median age 73 v 62 years; P =.02) patients with no other pretreatment clinical or laboratory features predicting occurrence of these events. The overall response rate was 45% (3% CR, 42% PR; 95% CI 28% to 64%). Median response duration for these 15 patients was 10 months (95% CI, 6.8-13.2; range, 3 to 17+). CONCLUSION: Rituximab administered thrice weekly for 4 weeks demonstrates clinical efficacy and acceptable toxicity. Initial infusion-related events seem to be cytokine mediated and resolve by the third infusion making rapid administration possible. Future combination studies of rituximab with other therapies in CLL seem warranted.

Immunotherapy with rituximab during peripheral blood stem cell transplantation for non-Hodgkin's lymphoma.

Peripheral blood stem cell grafts from patients with lymphoma are often contaminated with neoplastic cells. Administration of a lymphoma-specific monoclonal antibody before collecting stem cells may be one way of reducing the contamination. Similarly, an antibody after transplantation at a time of minimal residual disease may increase the efficacy of the procedure. The objective of this study was to determine the safety of using rituximab as both an in vivo purging agent and a posttransplantation adjuvant. Eligible patients with lymphoma received 375 mg/m2 rituximab intravenously IV) on day 1, 2.5 g/m2 cyclophosphamide IV on day 4, and 10 microg/kg per day filgrastim starting on day 5 and continuing until completion of leukapheresis. Patients subsequently received a standard preparative regimen and then received 375 mg/m2 rituximab IV 7 days after platelet independence was achieved. Twenty-five patients (14 men, 11 women; median age, 51 years) were enrolled. Of the 25 patients, 23 received transplants after at least 2.0 x 10(6) CD34+ cells/kg were harvested. As determined with a sensitive polymerase chain reaction assay, 6 of 7 stem cell products tested were free of tumor contamination. All patients engrafted promptly, and the rituximab infusions were well tolerated. Transient neutropenia of uncertain etiology occurred in 6 patients a median of 99.5 days post-transplantation. An additional patient developed progressive pancytopenia. Rituximab used as an in vivo purging agent and adjuvant immunotherapy with peripheral blood stem cell transplantation for non-Hodgkin's lymphoma is a well-tolerated regimen. However, the ultimate determination of efficacy will require the results of ongoing studies.

Theophylline, pentostatin (Nipent), and chlorambucil: a dose-escalation study targeting intrinsic biologic resistance mechanisms in patients with relapsed lymphoproliferative disorders.

In spite of the chemosensitivity seen with the initial treatment of malignant lymphoid disorders, relapse is common and death most often occurs as a result of disease progression. This is related to a multitude of resistance mechanisms associated with the various lymphoproliferative disorders. As a result, therapies targeting intrinsic drug-resistance mechanisms are evolving and have become an active area of research. In vitro studies of human chronic lymphocytic leukemia cells incubated with theophylline, a phosphodiesterase inhibitor, resulted in downregulation of bcl-2 concomitant with induction of apoptosis. We describe the preclinical basis for a novel combination therapy involving pentostatin (Nipent; SuperGen, San Ramon, CA), chlorambucil, and theophylline in the treatment of patients with relapsed chronic lymphoproliferative disorders. An ongoing study based on such justification, which is currently accruing patients, is also described. Results from this trial appear promising, and a phase II study is now being planned.

Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli.

To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs. They bear a stable hairpin stem-loop on the 3' end left from rho-independent termination and are known to undergo processing by RNase E. A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3' end. In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated. This approximately 125-nt RNA proved to arise via RNase E cleavage from the 3'-terminal region of the mRNAs bearing the terminator. Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly. The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection. Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5' --> 3' wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment. The results place polyadenylation at a later rather than an initiating step of decay. They also provide a clear illustration of how stably structured RNA 3' ends act as barriers to 3' --> 5' exonucleolytic mRNA decay.

Pedicle screw instrumentation for unstable thoracolumbar fractures.

We evaluated the results of short-segment pedicle screw instrumentation in 54 patients with unstable thoracolumbar fractures. Follow-up averaged 25 months (range, 11 to 36 months); 42 patients completed the study. Kyphosis was corrected by an average of 7 degrees at surgery and loss of correction averaged 5 degrees at the end of follow-up. On computed tomography, canal compromise averaged 57% preoperatively and 33% postoperatively. Complications included nerve root irritation due to screw penetration (1/42), screw breakage (2/42), and screw bending (6/42). Solid fusion was achieved in all cases at an average of 3 months. Of the 31 patients with normal neurologic function, 24 (77%) were pain-free at follow-up and had returned to previous levels of activity. We conclude that short-segment fixation with posterolateral fusion is effective in the treatment of unstable thoracolumbar fractures; it prevents progression of kyphotic deformity and neurologic deterioration, results in a stable fusion, and preserves uninvolved motion segments above and below the fracture site.

Ossification of the posterior longitudinal ligament: a report of nine cases in non-Oriental patients.

Ossification of the posterior longitudinal ligament (OPLL) is a progressive disorder of the spine which may result in spinal cord compression and myelopathy. While prevalent among Japanese, its occurrence in non-Orientals has been infrequently reported. Nine patients with OPLL have been diagnosed and followed at the Emory Clinic Spine Center over a 5-year period. All of the patients had been misdiagnosed before presentation. Five of the nine had undergone a total of eight ineffective operations. Failure to distinguish OPLL from other more common causes of myelopathy can result in delayed or inappropriate treatment. Illustrative cases and radiographic studies are presented.

Nicotine on the revascularization of bone graft. An experimental study in rabbits.

STUDY DESIGN: In 24 rabbits, the authors transplanted autologous cancellous bone to the anterior chamber of the eye. Half of the rabbits received nicotine and half received placebo (albumin) from mini-osmotic pumps that were implanted subcutaneously. Revascularization of the bone graft was evaluated postoperatively using ophthalmology slit-lamp and fluorescein angiography, and after sacrifice using microvascular silicone injection and histology. OBJECTIVES: The hypothesis that nicotine inhibits the revascularization of bone graft because of its pharmacologic action on the microvasculature was tested. SUMMARY OF BACKGROUND DATA: Pseudoarthrosis after spinal fusion occurs more frequently in smokers as compared with nonsmokers. METHODS: Observations of the bone graft were made regarding the time after implantation when vessels within the graft were noted and the pattern of these vessels. Revascularization of the graft was graded based on the observed percent area of fluorescence after injection of fluorescein. Serum levels of nicotine were measured weekly. Colored silicone was injected at sacrifice to fix the vasculature of the bone graft. Histologic analysis of undecalcified sections was performed. RESULTS: Nicotine, as compared with placebo, was associated with delayed revascularization within the graft, a smaller percent area of revascularization, and a larger number of grafts showing necrosis. CONCLUSIONS: Nicotine inhibits, but does not prevent, the revascularization of cancellous bone grafts. Inhibition of early revascularization by nicotine is proposed as the pathophysiologic mechanism by which smoking may adversely affect the healing of spinal fusions.

Impact of non-CTCL dermatologic diagnoses and adjuvant therapies on cutaneous T-cell lymphoma patients treated with total skin electron beam radiation therapy.

PURPOSE: To evaluate the impact of pre-cutaneous T-cell lymphoma dermatologic diagnoses and adjuvant therapies on the relapse-free and overall survivals of patients treated with total skin electron beam therapy. METHODS AND MATERIALS: Between 1974 and 1990, 164 patients were evaluated by members of Yale University School of Medicine departments of Dermatology and Therapeutic Radiology and treated with total skin electron beam therapy to a total dose of 3600 cGy. Patients who achieved a clinical complete response were offered doxorubicin/cyclophosphamide chemotherapy, extracorporeal photopheresis, or no systemic adjuvant therapy. The effects of TNM stage, antecedent non-T-cell lymphoma dermatologic diagnoses, and systemic adjuvant therapies were analyzed for their impact on relapse-free and overall survival. RESULTS: In this cohort of patients, an antecedent dermatologic diagnosis of follicular mucinosis or lymphomatoid papulosis was significantly associated with a shorter relapse-free survival for T1 and T2 patients, while antecedent "non-specific" dermatitides were associated with a somewhat better relapse-free survival. When the impact of systemic adjuvant therapies was analyzed, neither systemic doxorubicin/cyclophosphamide chemotherapy nor systemic extracorporeal photopheresis were found to delay cutaneous relapse. CONCLUSION: Our results suggest that antecedent follicular mucinosis and lymphomatoid papulosis may be associated with short relapse-free survival in T1 and T2 patients treated with total skin electron beam therapy. They also imply that neither adjuvant chemotherapy nor extracorporeal photopheresis delay cutaneous relapse after total skin electron beam therapy.

MR artifacts after anterior cervical diskectomy and fusion: a cadaver study.

OBJECTIVE: To identify the source of metallic artifacts observed on cervical spine MR studies following anterior cervical diskectomy and fusion (ACD&F), preoperative and postoperative MR studies were done on cadavers using standard spin echo T1, T2, and GRASS imaging. MATERIALS AND METHODS: An ACD&F was performed on each cadaver at various levels using different combinations of drill burrs, curettes, and suction tips. RESULTS: Results showed that contact between the drill burr and suction tip produced the worst artifacts; however, artifacts still occurred in cases in which the suction tip was not introduced. CONCLUSION: This observation suggests that metal flakes from the curettes also produced artifacts. Image distortion was greater on GRASS imaging than on the spin echo imaging.

Idiopathic CD4+ lymphocyte deficiency. Report of an unusual case associated with atopic dermatitis and allergic contact dermatitis and review of the literature.

BACKGROUND: Idiopathic CD4+ lymphocyte deficiency is a newly described entity of apparently non-human immunodeficiency virus-associated helper T-cell depletion. The clinical spectrum continues to evolve, but, to date, it has included patients ranging from those with minimal symptoms to those who have died with acute opportunistic infections. Several individual cases have been previously reported, many with distinctive, primarily infectious, cutaneous manifestations. OBSERVATIONS: We describe a patient with idiopathic CD4+ lymphocyte deficiency distinguished by a unique clinical presentation of atopic dermatitis exacerbated by contact urticaria and allergic contact dermatitis. She has a persistent markedly diminished CD4+ lymphocyte count (absolute count less than 50), no serologic evidence of, or risk factors for, human immunodeficiency virus infection, and no history of opportunistic infections. CONCLUSIONS: We present a possible new cutaneous manifestation of idiopathic CD4+ lymphocyte disease and summarize the previously reported cases, with an emphasis on the cutaneous features.