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Background: The stem cell marker nestin recently has been shown to be expressed in nascent blood vessels in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice. Materials and methods: In the present study, we visualized by dual-color fluorescence imaging tumor angiogenesis in the ND-GFP transgenic nude mice after orthotopic transplantation of the MIA PaCa-2 human pancreatic cancer line expressing red fluorescent protein. Mice were treated with gemcitabine at 150 mg/kg/dose on days 3, 6, 10, and 13 after tumor implantation. At day 14, mice were sacrificed and mean nascent blood vessel density and tumor volume were calculated and compared to control mice. Results: Nestin was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that CD31 co-localized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. Gemcitabine significantly decreased the mean nascent blood vessel density in the tumor as well as decreased tumor volume. Conclusion: The dual-color model of the ND-GFP nude mouse orthotopically implanted with RFP-expressing pancreatic tumor cells enabled the simultaneous visualization and quantitation of tumor angiogenesis and tumor volume. These results demonstrated for the first time that gemcitabine is an inhibitor of angiogenesis as well as tumor growth in pancreatic cancer. The results have important implications for the clinical application of gemcitabine in this disease. [Copyright &y& Elsevier]

We described 790 patients with newly diagnosed multiple myeloma, including 224 (28.4%) standard risk (SR) patients without t(11;14), 99 (12.5%) patients with t(11;14)alone, 58 (7.3%) with t(11;14) + HR, and 409 (51.8%) in the high-risk cytogenetic abnormality (HRCA) group including t(4;14), t(14;16), t(14;20), C1A1 and/or del(17p) but without t(11;14), to evaluate the impact of t(11;14) in NDMM patients on response rate, response kinetics and survival. Our study showed that NDMM patients in the t(11;14)alone group had similar PFS (49.3 vs. 50.7 months; P = 0.392) and OS (112.4 vs. NR months; P = 0.982) as those in the SR group. However, the t(11;14)alone group exhibited a significantly poorer depth of response compared to the SR group, particularly with a lower MRD negativity rate (60.0% vs. 76.0%, P = 0.009). In the t(11;14)alone group, MRD status did not significantly impact PFS or OS, which was in contrast to the other groups. Response kinetics analyses showed that the t(11;14)alone group had a slower response rate than the other subgroups (t(11;14)alone vs. SR vs. HRCA: median time to MRD negativity = 9.19 vs. 4.25 vs. 4.27 months; P < 0.001). Our study showed that t(11;14)alone was characterized by survival comparable to standard risk cytogenetics despite exhibiting the slowest timing of response onset and lowest plateau of remission, which suggested a relatively indolent clinical course. [ABSTRACT FROM AUTHOR]

Treatment for patients with multiple myeloma has experienced rapid development and improvement in recent years; however, patients continue to experience relapse, and multiple myeloma remains largely incurable. B-cell maturation antigen (BCMA) has been widely recognized as a promising target for treatment of multiple myeloma due to its exclusive expression in B-cell linage cells and its critical role in the growth and survival of malignant plasma cells. Here, we introduce STI-8811, a BCMA-targeting antibody-drug conjugate (ADC) linked to an auristatin-derived duostatin payload via an enzymatically cleavable peptide linker, using our proprietary C-lock technology. STI-8811 exhibits target-specific binding activity and rapid internalization, leading to G2/M cell-cycle arrest, caspase 3/7 activation, and apoptosis in BCMA-expressing tumor cells in vitro. Soluble BCMA (sBCMA) is shed by multiple myeloma cells into the blood and increases with disease progression, competing for ADC binding and reducing its efficacy. We report enhanced cytotoxic activity in the presence of high levels of sBCMA compared with a belantamab mafodotin biosimilar (J6M0-mcMMAF). STI-8811 demonstrated greater in vivo activity than J6M0-mcMMAF in solid and disseminated multiple myeloma models, including tumor models with low BCMA expression and/or in large solid tumors representing soft-tissue plasmacytomas. In cynomolgus monkeys, STI-8811 was well tolerated, with toxicities consistent with other BCMA-targeting ADCs with auristatin payloads in clinical studies. STI-8811 has the potential to outperform current clinical candidates with lower toxicity and higher activity under conditions found in patients with advanced disease. Significance: STI-8811 is a BCMA-targeting ADC carrying a potent auristatin derivative. We report unique binding properties which maintain potent cytotoxic activity under sBCMA-high conditions that hinder the clinical efficacy of current BCMA-targeting ADC candidates. Beyond disseminated models of multiple myeloma, we observed efficacy in solid tumor models of plasmacytomas with low and heterogenous BCMA expressions at a magnitude and duration of response exceeding that of clinical comparators., (©2024 The Authors; Published by the American Association for Cancer Research.)

Residual normal plasma cells (NPCs), which compete with tumor plasma cells, play an important role in multiple myeloma. However, large-scale cohort studies investigating residual NPCs, especially at the minimal residual disease (MRD) phase, are currently lacking. In this study, we conducted a comprehensive investigation into the clinical significance of residual NPCs throughout the entire disease course in 1363 myeloma patients from the NICHE cohort (NCT04645199). Our results revealed that myeloma patients with high baseline NPCs ratio (≥5%) exhibited distinct indolent features, characterized by lower tumor burden, reduced frequencies of cytopenia, immunoparesis, and high-risk cytogenetics. Importantly, high residual NPCs ratio at diagnosis or relapse was independently associated with favorable survival. High absolute percentages of NPCs at undetectable MRD were related with superior clinical benefit and immune reconstitution. At MRD-positive phases, grouping based on NPCs ratio (<50%, 50-90%, ≥90%) demonstrated better risk stratification compared to residual tumor log levels. Based on the time-dependent NPCs ratio trend, we developed a dynamic MRD model that classifies patients into three groups with diverse longitudinal trends, leading to distinct prognoses. Collectively, residual NPCs serves not only as a valuable complementary biomarker for risk stratification but also provides valuable insights on reclassifications and kinetics of MRD., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Growing evidence suggests that gain or amplification [gain/amp(1q)] accumulates during disease progression of multiple myeloma (MM). Previous investigations have indicated that small gain/amp(1q) subclones present at the time of diagnosis may evolve into dominant clones upon MM relapse. However, the influence of a minor clone of gain/amp(1q) on MM survival, as well as the correlation between different clonal sizes of gain/amp(1q) and the chromosomal instability (CIN) of MM, remains poorly understood. In this study, we analyzed fluorescence in situ hybridization (FISH) results of 998 newly diagnosed MM (NDMM) patients. 513 patients were detected with gain/amp(1q) at diagnosis. Among these 513 patients, 55 had a minor clone (≤20%) of gain/amp(1q). Patients with a minor clone of gain/amp(1q) displayed similar survival outcomes compared to those without gain/amp(1q). Further analysis demonstrated patients with a minor clone of gain/amp(1q) exhibited a clonal architecture similar to those without gain/amp(1q). Lastly, our results showed a significant increase in the clonal size of the minor clone of gain/amp(1q), frequently observed in MM. These findings suggested that a minor clone of gain/amp(1q) might represent an earlier stage in the pathogenesis of gain/amp(1q) and propose a "two-step" process in the clonal size changes of gain/amp(1q) in MM., (© 2024. The Author(s).)

Prognostic significance of multiple immune antigens in multiple myeloma has been well established. However, a level of uncertainty remains regarding the intrinsic relationship between immunophenotypes and cytogenetic stability and precise risk stratification. To address these unresolved issues, we conducted a study involving 1389 patients enrolled in the National Longitudinal Cohort of Hematological Diseases in China (NCT04645199). Our results revealed that the correlation between antigen expression and cytogenetics is more prominent than cytopenia or organ dysfunction. Most immune antigens, apart from CD38, CD138, and CD81, exhibit significant associations with the incidence of at least one cytogenetic abnormality. In turn, we identified CD138-low/CD27-neg as specific adverse immunophenotypic profile, which remaining independent impact on progression-free survival (HR, 1.49; P = 0.007) and overall survival (HR, 1.77; P < 0.001) even in the context of cytogenetics. Importantly, CD138-low/CD27-neg profile was also associated with inferior survival after first relapse (P < 0.001). Moreover, the antigen expression profiles were not strictly similar when comparing diagnosis and relapse; in particular, the CD138-low/CD27-neg pattern was notably increased after disease progression (19.1 to 29.1%; P = 0.005). Overall, our study demonstrates that diverse immune profiles are strongly associated with cytogenetic stability, and a specific immunophenotype (CD138-low/CD27-neg) could effectively predict prognoses across different disease stages., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: We investigated both the clinical utilities and the prognostic impacts of the clonotypic peptide mass spectrometry (MS)-EasyM, a blood-based minimal residual disease (MRD) monitoring protocol in multiple myeloma., Experimental Design: A total of 447 sequential serum samples from 56 patients with multiple myeloma were analyzed using EasyM. Patient-specific M-protein peptides were sequenced from diagnostic samples; sequential samples were quantified by EasyM to monitor the M-protein. The performance of EasyM was compared with serum immunofixation electrophoresis (IFE), bone marrow multiparameter flow cytometry (MFC), and next-generation flow cytometry (NGF) detection. The optimal balance of EasyM sensitivity/specificity versus NGF (10-5 sensitivity) was determined and the prognostic impact of MS-MRD status was investigated., Results: Of the 447 serum samples detected and measured by EasyM, 397, 126, and 92 had time-matching results for comparison with serum IFE, MFC-MRD, and NGF-MRD, respectively. Using a dotp >0.9 as the MS-MRD positive, sensitivity was 99.6% versus IFE and 100.0% versus MFC and NGF. Using an MS negative cutoff informed by ROC analysis (<1.86% of that at diagnosis), EasyM sensitivity remained high versus IFE (88.3%), MFC (85.1%), and NGF (93.2%), whereas specificity increased to 90.4%, 55.8%, and 93.2%, respectively. In the multivariate analysis, older diagnostic age was an independent predictor for progression-free survival [PFS; high risk (HR), 3.15; 1.26-7.86], the best MS-MRD status (MS-MRD negative) was independent predictor for both PFS (HR, 0.25; 0.12-0.52) and overall survival (HR, 0.16; 0.06-0.40)., Conclusions: EasyM is a highly sensitive and minimal invasive method of MRD monitoring in multiple myeloma; MS-MRD had significant predictive ability for survival outcomes., (©2024 The Authors; Published by the American Association for Cancer Research.)

The deletion of chromosome 17p (del(17p)) is considered a crucial prognostic factor at the time of diagnosis in patients with multiple myeloma (MM). However, the impact of del(17p) on survival at different clonal sizes at relapse, as well as the patterns of clonal evolution between diagnosis and relapse and their prognostic value, has not been well described. To address these issues, we analyzed the interphase fluorescence in situ hybridization (iFISH) results of 995 newly diagnosed MM (NDMM) patients and 293 patients with MM at their first relapse. Among these patients, 197 had paired iFISH data at diagnosis and first relapse. Our analysis of paired iFISH revealed that a minor clone of del(17p) at relapse but not at diagnosis was associated with poor prognosis in MM (hazard ratio for median overall survival 1.64 vs. 1.44). Fifty-six and 12 patients developed one or more new cytogenetic abnormalities at relapse, mainly del(17p) and gain/amp(1q), respectively. We classified the patients into six groups based on the change patterns in the clonal size of del(17p) between the two time points. Patients who did not have del(17p) during follow-up showed the best outcomes, whereas those who acquired del(17p) during their disease course, experienced compromised survival (median overall survival: 61.3 vs. 49.4 months; hazard ratio =1.64; 95% confidence interval: 1.06-2.56; P<0.05). In conclusion, our data confirmed the adverse impact of a minor clone of del(17p) at relapse and highlighted the importance of designing optimal therapeutic strategies to eliminate high-risk cytogenetic abnormalities (clinicaltrials gov. identifier: NCT04645199).

Objective: Multiple myeloma is a highly heterogenous plasma cell malignancy, commonly seen in older patients. Age is one of the important prognostic factors. However, nearly all the prognostic staging systems are based on clinical trials, where patients were relatively fit and young. It is unknown how the presence of biochemical or cytogenetic prognostic factors and their risk weights changes with older age. To further investigate this question, we retrospectively analyzed the data from a consecutive cohort of patients treated with either bortezomib or thalidomide-based therapy., Methods: This retrospective study was carried out on a cohort of 1125 newly diagnosed multiple myeloma patients, from January 2008 to December 2019. Patients received bortezomib or thalidomide-based induction and maintenance therapy. Patients accepted hematopoietic stem cell transplantation if eligible. Statistical analysis was conducted by Stata/MP 16.0 and SPSS 26.0., Results: With age increasing, the proportion of patients with ISS 3, performance status score ≥2, and the incidence rate of gain(1q) significantly increased. We also found that ISS became less important in older patients. However, cytogenetic abnormalities exerted a consistently adverse impact on survival, both in young and old patients. Older patients had an inferior outcome than their young counterparts. All patients in our cohort benefitted more from bortezomib than thalidomide-based induction therapy, except for patients ≥71 years old., Conclusions: ISS may lose prognostic value in patients ≥71 years old. Older patients had an inferior outcome and needed more effective and less toxic treatment. Plain Language Summary Multiple myeloma is a type of blood cancer commonly seen in older people. To treat this disease, genetic abnormality, the poor physical status of patients and the abundance of tumor cells are the main difficulties. We often draw these conclusions from clinical trials. However, clinical trials always enrolled relatively younger patients, so the presence and significance of these factors may vary from clinical trials to the real world. We conducted the study to find out the real risk in both young and old patients. We found that older patients were more likely to have anemia, poor nutritional status and renal function. We also found older patients had more risk of relapse, progression or death than young patients. Frail physical status is the key obstacle to treating older patients, and tumor burden no longer impacts the outcome of these people. Bortezomib is a powerful drug to treat this disease, but patients ≥71 years old had less benefit than younger ones. More studies should focus on older or frail patients as these patients need more effective and less toxic treatment.

Autologous stem cell transplantation (ASCT) is the standard therapy for patients with transplant-eligible multiple myeloma (TEMM). However, the ideal depth of response required before ASCT and the impact of residual tumor cells in the stem cell collection (SCC) on survival remains unclear. Here we collected data of 89 patients with TEMM undergoing ASCT and analyzed the minimal residual disease of SCC (cMRD) and bone marrow (BM) (mMRD) before transplantation. Before ASCT, 31.5% and 76.4% of patients achieved MRD negativity in BM and SCC, respectively. Tumor cells were less in SCC samples than that in BM samples. Neoplastic cells in SCC could be observed in patients with different responses after induction therapy, and there were no significant differences in the percentage and level of cMRD among these subgroups (P > 0.05). No correlation was found between the cMRD status and the response patients achieved after ASCT (P > 0.05). The median follow-up was 26.8 months. mMRD negativity before ASCT was associated with longer PFS (55.9 vs. 27.1 months; P = 0.009) but not OS (not reached vs. 58.9 months; P = 0.115). Patients with different cMRD statuses before ASCT experienced similar PFS (40.5 vs. 76.4 months for negativity vs. positivity; P = 0.685) and OS (not reached vs. 58.8 months for negativity vs. positivity; P = 0.889). These results suggested that detectable cMRD does not significantly predict the inferior post-ASCT response or shorter survival, and patients are eligible to undergo ASCT upon achieving partial response., (© 2023. The Author(s).)

Attaining undetectable minimal residual disease (MRD) is the current therapeutic goal for multiple myeloma. But there is a current lack of data regarding the clinical benefit of autologous stem cell transplantation (ASCT) for patients with myeloma achieving early MRD-negative status after induction treatment, in addition to the interaction of longitudinal MRD status with ASCT. The current study included 407 patients with transplant-eligible multiple myeloma with available MRD status from the National Longitudinal Cohort of Hematological Diseases in China (NCT04645199), of whom 147 (34.4%) achieved early undetectable MRD and 182 (44.7%) received ASCT. Early MRD-negative status was associated with a lower risk of disease progression [HR = 0.447; 95% confidence interval (CI), 0.333-0.600; P < 0.001] and death (HR = 0.473; 95% CI, 0.320-0.700; P < 0.001). Of note, patients who achieved undetectable MRD early still benefitted from ASCT, with a remarkable improvement in the median MRD-negative duration (33.5-58.0 months, P < 0.001), progression-free survival (PFS; 46.0-88.3 months, P < 0.001), and overall survival (OS; 76.4 months to not reached, P = 0.003). These clinical benefits were more pronounced in patients with aggressive features (high-risk cytogenetic abnormalities or high tumor burden) compared with standard-risk patients. Similar results were observed in patients with detectable MRD after induction treatment. In addition, we identified four MRD-status transformation patterns following ASCT, which were strongly correlated with diverse survival outcomes ( P < 0.001). Our study revealed the enhanced clinical significance of ASCT in patients with transplant-eligible myeloma, regardless of early MRD status, particularly for high-risk patients. Subsequent prospective trials are essential to validate these observations., Significance: Achieving and maintaining undetectable MRD is the current treatment goal for multiple myeloma. Our results emphasized the remarkable clinical benefit of ASCT on MRD-negative duration, PFS, and OS in patients with multiple myeloma regardless of early MRD status. These favorable impacts were more evident in patients with aggressive features. Importantly, dynamic MRD monitoring among ASCT could facilitate personalized stratification of therapeutic approaches., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Background: Solid tumors are becoming prevalent affecting both old and young populations. Numerous solid tumors are associated with high cMET expression. The complexity of solid tumors combined with the highly interconnected nature of the cMET/HGF pathway with other cellular pathways make the pursuit of finding an effective treatment extremely challenging. The current standard of care for these malignancies is mostly small molecule-based chemotherapy. Antibody-based therapeutics as well as antibody drug conjugates are promising emerging classes against cMET-overexpressing solid tumors., Research Design and Methods: In this study, we described the design, synthesis, in vitro and in vivo characterization of cMET-targeting Fab drug conjugates (FDCs) as an alternative therapeutic strategy. The format is comprised of a Fab conjugated to a potent cytotoxic drug via a cleavable linker employing lysine-based and cysteine-based conjugation chemistries., Results: We found that the FDCs have potent anti-tumor efficacies in cancer cells with elevated overexpression of cMET. Moreover, they demonstrated a remarkable anti-tumor effect in a human gastric xenograft mouse model., Conclusions: The FDC format has the potential to overcome some of the challenges presented by the other classes of therapeutics. This study highlights the promise of antibody fragment-based drug conjugate formats for the treatment of solid tumors.

Human scalp skin with abundant hair follicles in various stages of the hair growth cycle was histocultured for up to 40 days on Gelfoam ® at the air/liquid interface. The anagen hair follicles within the histoculture scalp skin produced growing hair shafts. Hair follicles could continue their cycle in histoculture; for example, apparent spontaneous catagen induction was observed both histologically and by the actual regression of the hair follicle. In addition, vellus follicles were shown to be viable at day 40 after initiation of culture. Follicle keratinocytes continued to incorporate [ 3 H]thymidine for up to several weeks after shaft elongation had ceased. Intensive hair growth was observed in the pieces of shaved mouse skin histocultured on Gelfoam ® . Isolated human and mouse hair follicles also produced growing hair shafts. By day 63 in histoculture of mouse hair follicles, the number of hair follicle-associated pluripotent (HAP) stem cells increased significantly and the follicles were intact. Gelfoam ® histoculture of skin demonstrated that the hair follicle cells are the most sensitive to doxorubicin which prevented hair growth, thereby mimicking chemotherapy-induced alopecia in Gelfoam ® histoculture.

Cells expressing the stem cell marker, nestin, were selectively labeled in transgenic mice by placing green fluorescent protein (GFP) under the control of the nestin promoter in transgenic mice. In these transgenic mice, neural and other stem cells brightly expressed GFP. The mice were termed nestin-driven GFP (ND-GFP) mice. During early anagen or growth phase of the hair follicle, ND-GFP appeared in the permanent upper hair follicle immediately below the sebaceous glands in the follicle bulge. The relatively small, oval-shaped, nestin-expressing cells in the bulge area surrounded the hair shaft and were interconnected by short dendrites. The location of the nestin-expressing cells in the hair follicle varied with the hair cycle. During telogen or resting phase and in early anagen, the GFP-positive cells are mainly in the bulge area. However, in mid- and late-anagen, the GFP-expressing cells were located in the upper outer-root sheath as well as in the bulge area. The expression of the unique protein, nestin, in both neural stem cells and hair follicle stem cells, which suggested their relationship. The ND-GFP hair follicle stem cells were later termed hair-follicle-associated pluripotent (HAP) stem cells.

Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair-follicle-associated pluripotent (HAP) stem cells. Cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells are described in this chapter. Intact hair follicles from green fluorescent protein (GFP) transgenic mice were cryopreserved by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in DMEM with fetal bovine serum (FBS). After 4 weeks culture, cells from the upper part of the hair follicles grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week culture, the growing cells formed hair spheres, each containing approximately 1 × 10(2) HAP stem cells. The hair spheres contained cells which could differentiate to neurons, glial cells, and other cell types. The formation of hair spheres by the thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. These results suggest that the cryopreservation of the whole hair follicle is an effective way to store HAP stem cells for personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use.

Acne is a very widespread cosmesis problem. Isotretinoin, a synthetic oral retinoid is used to treat acne, which is androgen dependent. Numerous side-effects occur from this treatment. 5-alpha-Reductase plays a critical role in normal and pathological androgen-dependent processes. We have taken the approach to develop a selective, effective, topically-applied 5-alpha-reductase inhibitor to modify unwanted or pathological processes in the pilosebaceous unit such as acne. Toward this goal, we have previously developed a selective liposome hair follicle targeting system. We demonstrate in this report that the 5-alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5alpha-androstane-17beta-carboxamide (4-MA) incorporated into liposomes induces apoptosis and inhibits growth of the dihydrotestosterone (DHT)-dependent hamster flank organ sebaceous gland. We have compared topical application of liposome 4-MA and solvent-formulated 4-MA and observed selective efficacy of topical application of liposome 4-MA by the reduction of size and induction of apoptosis only in the treated hamster flank organ. Apoptosis induced by liposome 4-MA in the treated flank organ sebaceous gland cells was observed both by assays for DNA fragments (transferase deoxytidyl uridine end labeling) and by observation of condensed and fragmented nuclei. When 4-MA was topically applied formulated in ethanol and glycerol without liposomes, the selective efficacy was lost. Liposome 4-MA did not significantly affect prostate weight, testosterone/DHT ratios or bodyweight gain compared to controls indicating safety as well as efficacy of topical application of liposome 4-MA for pathological processes such as acne.

CVX-22 is a CovX-Body, produced by covalently attaching a thrombospondin-1 (TSP-1) type 1 repeat peptide mimetic to a humanized IgG1 molecule. To dissect the antiangiogenic mechanism of CVX-22, the numbers and proliferative status of defined tumor endothelial cell (TEC) subsets from the B16 and C32 melanoma models were examined. CVX-22 treatment reduced the numbers of activated, vascular endothelial growth factor receptor 2 (VEGFR2)-positive TECs. Because the vast majority of mitotically active TECs reside in the VEGFR2 subset, a reduction in numbers of this compartment resulted in an 82% overall decrease in BrdU labeling of TEC. However, the rate of proliferation and VEGFR2 receptor density of this VEGFR2-positive subpopulation were unaffected. Instead, CVX-22 induced endothelial cell apoptosis both in vitro and in vivo, indicating that CVX-22 acts by selective deletion of activated, VEGFR2-positive TEC. The overrepresentation of activated cells in sites of tumor angiogenesis may confer a unique specificity of CVX-22 for tumor vasculature.

The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, also is expressed in follicle stem cells and their immediate, differentiated progeny. The fluorescent protein GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34 but negative for keratinocyte marker keratin 15, suggesting their relatively undifferentiated state. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In vivo studies show the nestin-driven GFP hair follicle stem cells can differentiate into blood vessels and neural tissue after transplantation to the subcutis of nude mice. Equivalent hair follicle stem cells derived from transgenic mice with beta-actin-driven GFP implanted into the gap region of a severed sciatic nerve greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells, which are known to support neuron regrowth. Function of the rejoined sciatic nerve was measured by contraction of the gastrocnemius muscle upon electrical stimulation. After severing the tibial nerve and subsequent transplantation of hair follicle stem cells, walking print length and intermediate toe spread significantly recovered, indicating that the transplanted mice recovered the ability to walk normally. These results suggest that hair follicle stem cells provide an important, accessible, autologous source of adult stem cells for regenerative medicine.