Bone Marrow Implantation Research Articles

Autologous Bone Marrow Transplantation and Hyperbaric Oxygen Therapy for Patients With Thromboangiitis Obliterans

Many patients suffering from severe ischemic limb disease inevitably experience amputation, despite intensive therapies. Sympathectomy and hyperbaric oxygen therapy are optional therapies for patients with peripheral circulation disorders. Recently, several clinical studies have established that implantation of bone marrow-mononuclear cells into ischemic limbs increases collateral vessel formation. In the present study, autologous implantation of bone marrow-mononuclear cells was prescribed to 7 patients with ischemic limbs because of peripheral arterial disease. Although the extent of the improvement was not consistent among the 7 cases, all of the patients experienced some improvement in their symptoms. Transcutaneous oxygen partial pressure measured in a hyperbaric chamber increased in 5 patients. No side effects were observed. In conclusion, combined use of autologous bone marrow transplantation and hyperbaric oxygen therapy may be safe and effective for achievement of therapeutic angiogenesis.

Serum erythropoietin level as a marker of limb ischemia

Bone marrow implantation (BMI) has been utilized for the treatment of limb ischemia, however, serum markers have not yet been reported to express the degree of limb ischemia. We analyzed the serum levels of several cytokines including erythropoietin (EPO) in the treated legs and the contralateral ones in 11 patients with limb ischemia treated with BMI. The EPO level in the pre-treated legs in the 5 patients with arteriosclerosis obliterans revealed a good correlation with ankle–brachial pressure index. The EPO level, but not the levels of TNF-α, VEGF, and bFGF in the pre-treated legs was significantly higher than that in the contralateral legs in the 11 patients, and the EPO level decreased in 4 weeks after BMI. The serum EPO level may express the degree of limb ischemia presumably through the reactive production of EPO in ischemic tissue.

Local implantation of autologous mononuclear cells from bone marrow and peripheral blood for treatment of ischaemic digits in patients with connective tissue diseases

CD34-positive bone marrow mononuclear cells (MNCs) have been successfully used for regeneration of small arteries in Buerger's disease. The objective of this study is to examine the angiogenetic potential of autologous MNCs from bone marrow and peripheral blood implanted into the ischaemic digits from patients with connective tissue diseases. Three patients with systemic sclerosis, two with mixed connective tissue disease, and one with CREST syndrome were enrolled who had painful ischaemic digits with necrosis refractory to several vasodilators including intravenous prostaglandins. MNCs obtained from 7 ml/kg bone marrow blood and 400 ml peripheral blood were implanted into 20 different sites in palms and/or soles. The study was performed open-labelled. Pain in the numeric rating scale improved remarkably up to 1 month after implantation of bone marrow or peripheral MNCs to the same extent, although no significant differences were found in transcutaneous oxygen pressure and thermogram before and after the implantation. Bone marrow MNCs increased blood flow of the hand determined by intra-arterial digital subtraction angiography, while peripheral MNCs did not. Implantation of autologous MNCs from peripheral and bone marrow into the ischaemic digits was so effective in pain-relief and more clinical trials would be warranted to see whether this could be a new treatment modality for angiogenesis in connective tissue diseases as in Buerger's disease.

骨髄細胞・多孔性β‐ＴＣＰブロック複合体による骨形成に関する組織学的検討

骨髄細胞・多孔性β‐ＴＣＰブロック複合体による骨形成に関する組織学的検討

5.7 Chondrocytes versus fresh bone marrow implantation for cartilage repair - 3 year follow-up.

5.7 Chondrocytes versus fresh bone marrow implantation for cartilage repair - 3 year follow-up.

Morphological Transformation in Esophageal Submucosa by Bone Marrow Cells: Esophageal Implantation under External Esophageal Perfusion

Accumulating clinical and experimental studies indicate that Barrett's esophagus might arise through multipotential stem cells under the stress of gastroesophageal reflux. Previously, we have presented a novel external pump perfusion rat model and demonstrated that perfusion with both acid and bile can induce severe esophagitis in 1 week with a similarly pathological change seen in humans. The aim of this study was to investigate the histological changes of esophagus after bone marrow cell engraftment with bile and acid perfusion. The external pump perfusion procedure involved implantation of a microosmotic pump for esophageal perfusion. Bone marrow cells were obtained by flushing of the femur marrow, and the cell suspension was injected between the esophageal muscular and inner mucosa layer. Histological changes were determined after 4 weeks of perfusion. Proliferating cell nuclear antigen, 8-hydroxy-deoxyguanosine, manganese superoxide dismutase, and apoptosis were measured by immunohistochemical staining and TUNEL assay, respectively. Severe esophagitis was seen in both acid and bile perfusion. Bone marrow engraftment and potentiation was seen in both the acid and bile perfusion, when compared to saline controls. Glandular-like cells in submucosa, consistent with intestinal metaplasia, confirmed by Alcin Blue-PAS staining were observed after bone marrow esophageal implantation along with bile perfusion, but not with acid perfusion and controls. Bone marrow implantation in conjunction with esophageal reflux injury contributes to abnormal histological changes consistent with early Barrett's esophageal changes. Engrafted bone marrow cells proliferate under oxidative stress conditions with bile perfusion.

Bone tissue engineering using foetal cell therapy.

Different cell sources for bone tissue engineering are reviewed. In particular, adult cell source strategies have been based on the implantation of unfractionated fresh bone marrow; purified, culture expanded mesenchymal stem cells, differentiated osteoblasts, or cells that have been modified genetically to express rhBMP. Several limiting factors are mentioned for these strategies such as low number of available cells or possible immunological reaction of the host. Foetal bone cells are presented as an alternative solution and review of actual treatments using these cells is presented. Finally, foetal cells used specifically for bone tissue engineering are characterised and potentially interesting therapeutic options are proposed.

Bone tissue engineering using foetal cell therapy.

Different cell sources for bone tissue engineering are reviewed. In particular, adult cell source strategies have been based on the implantation of unfractionated fresh bone marrow; purified, culture expanded mesenchymal stem cells, differentiated osteoblasts, or cells that have been modified genetically to express rhBMP. Several limiting factors are mentioned for these strategies such as low number of available cells or possible immunological reaction of the host. Foetal bone cells are presented as an alternative solution and review of actual treatments using these cells is presented. Finally, foetal cells used specifically for bone tissue engineering are characterised and potentially interesting therapeutic options are proposed.

The Prostacyclin Analog Beraprost Sodium Augments the Efficacy of Therapeutic Angiogenesis Induced by Autologous Bone Marrow Cells

Implantation of autologous bone marrow (BM) mononuclear cells (MNCs) has been shown to augment neovascular formation in ischemic tissues in experimental animals and in humans. Prostaglandin derivatives improve the symptoms of patients with critical limb ischemia, possibly by their vasodilating and antiplatelet actions. We therefore examined whether therapeutic angiogenesis by implantation of autologous BM-MNCs would be enhanced by beraprost sodium (BPS), using a rabbit ischemic hindlimb model. Ischemia was induced by surgical resection of the left femoral artery. Twenty-five New Zealand white rabbits were divided into four groups. The first group (BM group, n = 4) received autologous BM-MNCs (2 x 10(6)/animal) implanted into the ischemic tissue 1 week after limb ischemia. The second group (BM+BPS group, n = 8) received BPS injected into the dorsal skin (300 microg/kg daily) starting 1 week before limb surgery. This group received BM-MNC implantation 1 week after surgery. Daily injection of BPS was continued until the end of the protocol. The third group (BPS group, n = 8) received BPS injected into the dorsal skin (600 microg/kg daily) starting 1 week before limb surgery. The fourth group received saline as a control (n = 4). The extent of angiogenesis in the ischemic hindlimb was assessed using the angiographic score (AS), ischemic/normal limb calf blood pressure ratio (CBPR), and tissue capillary density. Four weeks after limb ischemia, the ischemic/normal CBPR was highest in the BM+BPS group, followed by the BPS, BM, and control groups (0.56 +/- 0.16, 0.51 +/- 0.25, 0.44 +/- 0.15, and 0.30 +/- 0.10, respectively). The AS was also the greatest in the BM+BP group, followed by the BM, BP, and S group (1.63 +/-0.21, 1.31 +/- 0.25, 1.26 +/- 0.21 and 0.80 +/- 0.10, respectively). The TCD was greatest in the BM+BP group, followed in by the BM, BP, and S group? (46 +/- 4.1, 34 +/- 0.7, 33 +/- 6.9, and 19 +/- 1.8 per field, respectively). BP treatment is an effective means to enhance the efficacy of therapeutic angiogenesis induced by autologous BM-MNCs implantation in ischemic hindlimb tissues.

Therapeutic angiogenesis by bone marrow implantation for critical hand ischemia in patients with peripheral arterial disease: a pilot study

ABSTRACTObjective: Implantation of bone marrow mononuclear cells (BM-MNCs), including endothelial progenitor cells, into ischemic lower limbs has been shown to improve symptoms in patients with peripheral arterial diseases (PAD). This study investigated whether BM-MNC implantation (BMI) is also effective for the ischemic hands of these patients.Methods: Seven PAD patients with hand ischemia were enrolled: six patients had thromboangiitis obliterans and one had collagen disease. All seven had symptoms involving either resting pain or non-healing ischemic ulcers of the hand. Approximately 600 mL of MNCs were separated from BM and concentrated to a final volume of 40–50 mL, which were injected into ischemic hands. Ischemic status was evaluated by measuring the digital/brachial pressure index (DBI), visual analog pain scale, and the healing of ulcers before and 6 months after BMI.Results: The mean number of implanted MNCs, CD34-positive cells, and CD34,133-positive cells was 3.67 ± 0.53 × 109, 4.94 ± 2.45 × 107, and 2.52 ± 1.57 × 107, respectively. Mean DBI in those patients was 0.15 ± 0.30 before BMI and significantly increased to 0.67 ± 0.19 at 6 months after BMI ( p = 0.004). All patients also showed improvement of pain scale and ischemic ulcers. There was no significant correlation between the number of implanted cells and improvement in the degree of DBI or the pain scale.Conclusion: Autologous BMI could be a promising and safe method of therapeutic angiogenesis for critical hand ischemia in PAD patients.

The use of percutaneous autologous bone marrow transplantation in nonunion and avascular necrosis of bone

During the development of normal bone in the young child, osteoblasts and then haematopoietic stem cells progressively colonise the cartilaginous matrix, resulting in its ossification to bone matrix.[1][1] It is at this time that red bone marrow appears in the skeleton, starting distally in the foot

Combination Therapy with Interleukin-6 Receptor Superantagonist Sant7 and Dexamethasone Induces Antitumor Effects in a Novel SCID-huIn vivoModel of Human Multiple Myeloma

Interleukin-6 (IL-6) protects multiple myeloma cells against apoptosis induced by glucocorticoids. Here, we investigated whether inhibition of the IL-6 signaling pathway by the IL-6 receptor superantagonist Sant7 enhances the in vivo antitumor effects of dexamethasone on the IL-6-dependent multiple myeloma cell line INA-6. For this purpose, we used a novel murine model of human multiple myeloma in which IL-6-dependent INA-6 multiple myeloma cells were directly injected into human bone marrow implants in severe combined immunodeficient (SCID) mice (SCID-hu). The effect of in vivo drug treatments on multiple myeloma cell growth was monitored by serial determinations of serum levels of soluble IL-6 receptor (shuIL-6R), which is released by INA-6 cells and served as a marker of tumor growth. In SCID-hu mice engrafted with INA-6 cells, treatment with either Sant7 or dexamethasone alone did not induce significant reduction in serum shuIL-6R levels. In contrast, the combination of Sant7 with dexamethasone resulted in a synergistic reduction in serum shuIL-6R levels after 6 consecutive days of treatment. Gene expression profiling of INA-6 cells showed down-regulation of proliferation/maintenance and cell cycle control genes, as well as up-regulation of apoptotic genes in multiple myeloma cells triggered by Sant7 and dexamethasone combination. In vitro colony assays showed inhibition of myeloid and erythroid colonies from normal human CD34(+) progenitors in response to dexamethasone, whereas Sant7 neither inhibited colony growth nor potentiated the inhibitory effect of dexamethasone. Taken together, these results indicate that inhibition of IL-6 signaling by Sant7 significantly potentiates the therapeutic action of dexamethasone against multiple myeloma cells, providing the preclinical rationale for clinical trials of Sant7 in combination with dexamethasone to improve patient outcome in multiple myeloma.

Can a bone marrow cell contribute to organ regeneration? In vivo analysis using transgenic rats with reporter genes

Although implantation of multipotent bone marrow–derived stem cells represents an attractive new cell therapy to repair damaged tissues, recent reports have raised serious concerns over the feasibility of using stem cells deriving from the bone marrow to promote cell transdifferentiation. We established transgenic (Tg) rats with reporter genes as specific molecular tags to examine the effect of bone marrow cells (BMCs) on transdifferentiation into tissues/organs. To monitor transdifferentiation events of locally transplanted BMCs into hepatocytes or capillary endothelial cells, a liver injury model and an ischemic hind-limb model were developed in rats. To test the ability of circulating bone marrow–derived cells to give rise to myocytes after skeletal muscle injury, we used a bone marrow cell transplantation model from Tg rats, which showed ubiquitous expression of β-galactosidase (lacZ), into lethally irradiated non-Tg rats. Our results show that there was little transdifferentiation of BMCs into the targeted cells in these tissue injury models. However, in the ischemic hind-limb model, laser Doppler imaging and histologic analysis showed that both implantation of BMCs and treatment with microspheres incorporating basic fibroblast-like growth factor (bFGF), which enables the release of bFGF at the site of action over a period of time, effectively induced angiogenesis. In conclusion, rat BMCs with specific marker genes could be a useful tool for detecting transdifferentiation events in vivo.

Effect of G-CSF and Intramyocardial Injection of Bone Marrow Cells on Cardiac Function in Mouse Model of Myocardial Infarction: A Word for Neo-Vasculogenesis.

Recent studies suggest that primitive stem cells derived from bone marrow (BM) possess greater functional plasticity that was expected previously. It has been shown that bone marrow stem cells (BMSC) promote repairing mechanisms within myocardium following ischemia/reperfusion models of myocardial infarction (MI). Although it remains unclear whether BMSC transdifferentiate into or just fuse with cardiomyocytes, hemodynamic improvement after intramyocardial BMSC injection as well as after G-CSF injection has been demonstrated. Here, we investigated the contribution of BMSC versus G-CSF administration in myocardial repair following MI. Ten weeks old C57BL/6J mice were irradiated and transplanted with green fluorescent protein (GFP) positive bone marrow cells. Three months later, these mice underwent ligation of left anterior descending branch (LAD) of coronary artery and subsequently divided into three groups. One group (n=7) received G-CSF administration at 200ug/kg for 10 consecutive days. Another group (n=9) was injected with GFP+ marrow cells directly into ischemic heart. The third group was held as control (n=7). One month after coronary ligation we found significant improvement in cardiac function determined as a cardiac output, maximum power and dP/dt, in the G-CSF group compared to control. We evaluated the phenotype of GFP+ cells within myocardium in each treatment group by 488 nm laser-scanning confocal miscroscopy (of whole heart and slides) 35 days after LAD ligation. We found no evidence of myocardial transdifferentiation or cardiomyocyte cell fusion. Instead GFP+ capillaries were present and exclusively located in infarct border zones in both the G-CSF and bone marrow implantation groups, confirmed by anti-factor VIII staining. G-CSF administration and to a lesser extent marrow injection resulted in improved post infarct cardiac function indices. This beneficial effect is not due to transdifferentiation but could be explained by marrow injected or G-CSF mobilized endothelial progenitor cells (EPC) and/or cytokine mediated neo-vasculogenesis.

Depletion of alloantigen-primed lymphocytes overcomes resistance to allogeneic bone marrow in mildly conditioned recipients

Objective: Successful implantation of allogeneic bone marrow (BM) cells after nonmyeloablative conditioning would allow to compensate for the inadequate supply of compatible grafts and to reduce mortality of graft-vs.-host disease (GVHD). Recently, we proposed to facilitate engraftment of mismatched BM by conditioning for alloantigen-primed lymphocyte depletion (APLD) with cyclophosphamide (CY). Here we summarize the experimental results obtained by this approach. Materials and methods: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY (day 1) to deplete alloantigen-primed lymphocytes, and given a second C57BL/6 BM transplant (day 2) for engraftment. Recipients were repeatedly tested for chimerism in the blood and followed for GVHD and survival. The protocol was also tested for inducing tolerance to donor tissue and organ allografts, and for treatment of leukemia, breast cancer, and autoimmune diabetes in NOD mice. Results: APLD by 200 mg/kg CY provided engraftment of allogeneic BM from the same donor in 100% mildly irradiated recipients. Eighty percent chimeras remained GVHD-free more 200 days. All chimeras accepted permanently donor skin grafts and donor hematopoietic stromal progenitors. Allogeneic BM transplantation (BMT) after APLD had a strong therapeutic potential in BALB/c mice harboring malignant cells and in autoimmune NOD recipients. Tolerance-inducing CY dose could be reduced to 100 mg/kg. Conditioning for APLD resulted in engraftment of allogeneic BM after a significantly lower radiation dose than treatment with radiation and CY alone. Conclusion: Our results demonstrate that conditioning for APLD has a definite advantage over general immunosuppression with CY and radiation therapy.

Clinical Application of Bone Marrow Implantation in Patients With Arteriosclerosis Obliterans, and the Association Between Efficacy and the Number of Implanted Bone Marrow Cells

There have been a number of recent reports on the use of autologous bone marrow implantation (BMI) in the treatment of peripheral arterial disease, with a clinical response rate of approximately 70%. However, the factors that influence efficacy have not yet been clarified. We have analyzed the relationship between the number of implanted bone marrow cells and the clinical efficacy of BMI. Eight patients with arteriosclerosis obliterans were treated with BMI. Bone marrow was aspirated from the ilium (500-1,000 ml), the mononuclear cells were separated and then were implanted. The clinical effectiveness of BMI was evaluated by assessing changes in the ankle-brachial pressure index (ABI) and the transcutaneous oxygen pressure (TcO2) between the pre-treatment baseline, with follow-up testing at 4 weeks. These changes were defined as DeltaABI and DeltaTcO2. The mean number of CD34-positive cells was 1.04+/-0.60 x10(6) /kg body weight. There was a strong correlation between the number of CD34-positive cells and DeltaABI (r=0.754, p=0.028). It is likely that the number of implanted CD34-positive cells is one of the primary factors that influence the clinical efficacy of BMI.

The induction of angiogenesis by the implantation of autologous bone marrow cells: A novel and simple therapeutic method

Background. Bone marrow contains many kinds of primitive cells that could differentiate to endothelial cells and secrete several growth factors. In the current study, we attempted to induce therapeutic angiogenesis by implanting autologous bone marrow cells (BMCs) and using a rat ischemic hind limb model. Methods. BMCs were prepared by removing red blood cells. A rat ischemic hind limb model was made by the ligation of the left femoral artery and its branches. BMCs were injected into 7 points of the ischemic muscles. To assess angiogenesis, a microangiogram, laser Doppler, and histologic evaluation were performed after the surgical procedure. Results. A microangiogram and histologic evaluation showed that angiogenesis was significantly induced in the ischemic hind limb by the implantation of BMCs. Laser Doppler imaging analysis showed that blood flow was significantly increased after implantation of BMCs. Some implanted BMCs were stained positively with CD31 and vascular endothelial-cadherin (VE-cadherin), which might have been incorporated into the vasculature. The condition of ischemia caused an elevation in the level of basic fibroblast growth factor in the ischemic muscle and also in interleukin-1β derived from the implanted BMCs, which might contribute to angiogenesis. Conclusion. These findings indicate that autologous bone marrow implantation may be a novel and simple method for inducing therapeutic angiogenesis. (Surgery 2001;130:44-54.)

Autologous Bone Marrow Implantation Induced Angiogenesis and Improved Deteriorated Exercise Capacity in a Rat Ischemic Hindlimb Model

Background. Bone marrow possesses endothelial progenitor cells that secrete several growth factors and can contribute to the formation of new capillaries. In the present study, we investigated the extent of angiogenesis induced by implantation of autologous bone marrow cells (BMCs) in a rat ischemic hindlimb model and studied whether the increased collateral vessels can improve deteriorated physical function.Materials and methods. Ischemic hindlimb was created by ligation of the femoral artery and its branches in Dark Agouti (DA) rats. BMCs (1 × 107) were injected percutaneously at six points into the gastrocnemius muscle. To assess angiogenesis, histologic evaluation and microangiography were performed at 2 weeks postligation. Severity of the ischemic insult was evaluated by measuring blood flow in the adductor and gastrocnemius muscles using nonradioactive colored microspheres and by determining the femoral arteriovenous oxygen difference (AVDO2) at 2 weeks postligation. Running time on a motor-driven treadmill was used to represent exercise capacity.Results. The histologic evaluation and microangiogram showed that the implanted BMCs induce angiogenesis. Blood flow to the adductor muscle on the treated side in the bone marrow cell implantation (BMI) group was significantly restored to 77.3 ± 19.3% of that of the normally perfused limb in comparison to that in control groups (P < 0.05). AVDO2 in the BMI group significantly decreased when compared with AVDO2 in control groups. Rats in the BMI group ran approximately 1.5 times longer than rats in control groups at 2 and 4 weeks postligation (P < 0.01).Conclusions. Implantation of autologous BMCs induced angiogenesis and improved deteriorated exercise capacity in our rat ischemic hindlimb model.

Reconstruction of the primate mandible with a combination graft of recombinant human bone morphogenetic protein-2 and bone marrow

Purpose: This study evaluated whether recombinant human bone morphogenetic protein-2 (rhBMP-2) can be used to regenerate a resected part of the mandible in a primate model. Materials and Methods: Segmental bone defects were created surgically in the mandible of Japanese monkeys. rhBMP-2 was suspended in a solution of polyglycolic co-lactic acid (PGLA) and lyophilized to make a BMP/PGLA complex. The rhBMP-2/PGLA complex and autogenous bone marrow in ratios of 3:0, 2.5:0.5, or 2:1 (vol:vol) were each implanted into the bone defects in 3 monkeys. Bone marrow or PGLA alone were each implanted in 1 monkey as a control. The animals were killed 16 weeks after surgery, followed by radiologic and histologic evaluation. Results: The implantation of bone marrow alone succeeded in reconstruction of the mandible, but the implantation of the rhBMP-2/PGLA complex showed only a small amount of bone formation. The combination graft of rhBMP-2/PGLA and bone marrow resulted in a greater degree of bone formation; especially the 2:1 combination showed the same result as only bone marrow implantation. Conclusion: The combination graft of rhBMP-2 and bone marrow, which requires only a small amount of bone marrow, was a reliable method for reconstruction of mandibular segmental defects in this animal model. © 2001 American Association of Oral and Maxillofacial Surgeons

In Response:

In our study, we classify CD34+Lin−Thy-1+ cells as human stem cells because the Thy-1+ subpopulation of CD34+ cells have self-renewal characteristics of human stem cells and are capable to generate all hematopoietic lineages. Effectively, primitive hematopoietic stem cells in normal human peripheral blood and bone marrow have been characterized [1Udomsakdi C. Lansdorp P.M. Hogge D.E. Reid D.S. Eaves A.C. Eaves C.J. Characterization of primitive hematopoietic cells in normal human peripheral blood.Blood. 1992; 80: 2513PubMed Google Scholar, 2Srour E.F. Brandt J.E. Briddell R.A. Leemhuis T. van Besien K. Hoffman R. Human CD34+ HLA-DR-bone marrow cells contain progenitors capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.Blood Cells. 1991; 17: 287PubMed Google Scholar, 3Baum C.M. Weissman I.L. Tsukamoto A.S. Buckle A.M. Peault B. Isolation of a candidate human hematopoietic stem cell population.Proc Natl Acad Sci U S A. 1992; 89: 2804Crossref PubMed Scopus (888) Google Scholar]. These cells are CD34+Lin−Thy-1+, constitute less than 0.05% of whole bone marrow, are capable of multilineage differentiation, including myeloid, B-, and T-cell lineages, and have extensive self-renewal capacity [3Baum C.M. Weissman I.L. Tsukamoto A.S. Buckle A.M. Peault B. Isolation of a candidate human hematopoietic stem cell population.Proc Natl Acad Sci U S A. 1992; 89: 2804Crossref PubMed Scopus (888) Google Scholar, 4Kyoizumi S. Baum C.M. Kaneshima H. McCune J.M. Yee E.J. Namikawa R. Implantation and maintenance of functional human bone marrow in SCID-hu mice.Blood. 1992; 79: 1704PubMed Google Scholar]. CD34+Lin−Thy-1+ cells resemble hematopoietic stem cells based on in vitro assays for long-term culture-initiating cells and in vivo assays for hematopoietic stem cells utilizing the SCID-hu bone assay system [5Craig W. Kay R. Cutler R.L. Lansdorp P.M. Expression of Thy-1 on human hematopoietic progenitor cells.J Exp Med. 1993; 177: 1331Crossref PubMed Scopus (421) Google Scholar] and SCID-hu thymus assay system [6Peault B. Weisman I.L. Baum C. McCune J.M. Tsukamoto A. Lymphoid reconstitution of the human fetal thymus in SCID mice with CD34+ Precursor cells.J Exp Med. 1991; 174: 1283Crossref PubMed Scopus (130) Google Scholar]. CD34+Lin−Thy-1+ cells have been shown to be able to fully reconstitute hematopoiesis in laboratory animals including non-human primates [7Donahue R.E. Kessler S.W. Kirby M.R. et al.Autologous bone marrow transplantation in non-human primates using a subpopulation of CD34+ cells expressing Thy-1.Blood. 1993; 82: 1175PubMed Google Scholar]. In humans, CD34+Lin−Thy-1+ cells can be mobilized in the peripheral blood using a high dose of cyclophosphamide and growth factor regimens and isolation in sufficient numbers to potentially permit hematologic reconstitution following myeloablative regimen. It was observed that the percentage of Thy-1+ in the CD 34+ population of successfully mobilized PBSC was regularly increased well above that seen in resting bone marrow, and it is far from clear what that means. The mobilization phenomenon may cause selective release of ontologically more primitive cells. Our study aimed at finding the minimal effective dose, which at that time was thought to be very low, and the median dose finally used in our study reflected the cell-dose-de-escalation design of the study with an actual median dose of 0.69. Therefore it was decided to base the analysis on a 0.8 × 106 CD+Lin−Thy-1+/kg threshold dose reported by Systemix in a more extensive analysis of a multidisease population. CD34+Lin−Thy-1+ cells are capable of producing fast and durable hematopoietic engraftment at cell doses greater than 0.8 × 106 CD34+ Thy−1+/kg, and the two patients (8 and 30) who experienced delayed engraftment developed sepsis, followed (for patient 30) by an hemolytic and uremic syndrome. Considering the number of CD34+ cells required for rapid engraftment, our results are comparable to those obtained after the infusion of 2.5 × 106 to 15 × 106 CD34+cells. Moreover, it has been demonstrated that the rapidity of hematopietic reconstitution was related to the number of CD34+ cells infused [8Ketterer N. Salles G. Raba M. et al.High CD 34+ cell count decrease hematologic toxicity of autologous peripheral blood progenitor cell transplantation.Blood. 1998; 91: 3148PubMed Google Scholar].